Gastrin releasing peptide compounds

ABSTRACT

New and improved compounds for use in diagnostic imaging or therapy having the formula M—N—O—P—G, wherein M is an optical label or a metal chelator (in the form complexed with a metal radionuclide or not), N—O—P is the linker, and G is the GRP receptor targeting peptide. Methods for imaging a patient and/or providing radiotherapy or phototherapy to a patient using the compounds of the invention are also provided. Methods and kits for preparing a diagnostic imaging agent from the compound is further provided. Methods and kits for preparing a radiotherapeutic agent are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of InternationalApplication PCT/US/2003/041328, filed Dec. 24, 2003, which is acontinuation-in-part application of U.S. application Ser. No. 10/341,577filed Jan. 13, 2003 now U.S. Pat. No. 7,226,577. All of theseapplications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to novel gastrin releasing peptide (GRP)compounds which are useful as diagnostic imaging agents orradiotherapeutic agents. These GRP compounds are labeled withradionuclides or labels detectable by in vivo light imaging and includethe use of novel linkers between the label and the targeting peptide,which provides for improved pharmacokinetics.

BACKGROUND OF THE INVENTION

The use of radiopharmaceuticals (e.g., diagnostic imaging agents,radiotherapeutic agents) to detect and treat cancer is well known. Inmore recent years, the discovery of site-directed radiopharmaceuticalsfor cancer detection and/or treatment has gained popularity andcontinues to grow as the medical profession better appreciates thespecificity, efficacy and utility of such compounds.

These newer radiopharmaceutical agents typically consist of a targetingagent connected to a metal chelator, which can be chelated to (e.g.,complexed with) a diagnostic metal radionuclide such as, for example,technetium or indium, or a therapeutic metal radionuclide such as, forexample, lutetium, yttrium, or rhenium. The role of the metal chelatoris to hold (i.e., chelate) the metal radionuclide as theradiopharmaceutical agent is delivered to the desired site. A metalchelator which does not bind strongly to the metal radionuclide wouldrender the radiopharmaceutical agent ineffective for its desired usesince the metal radionuclide would therefore not reach its desired site.Thus, further research and development led to the discovery of metalchelators, such as that reported in U.S. Pat. No. 5,662,885 to Pollaket. al., hereby incorporated by reference, which exhibited strongbinding affinity for metal radionuclides and the ability to conjugatewith the targeting agent. Subsequently, the concept of using a “spacer”to create a physical separation between the metal chelator and thetargeting agent was further introduced, for example in U.S. Pat. No.5,976,495 to Pollak et. al., hereby incorporated by reference.

The role of the targeting agent, by virtue of its affinity for certainbinding sites, is to direct the diagnostic agent, such as aradiopharmaceutical agent containing the metal radionuclide, to thedesired site for detection or treatment. Typically, the targeting agentmay include a protein, a peptide, or other macromolecule which exhibitsa specific affinity for a given receptor. Other known targeting agentsinclude monoclonal antibodies (MAbs), antibody fragments (F_(ab)'s and(F_(ab))₂'s), and receptor-avid peptides. Donald J. Buchsbaum, “CancerTherapy with Radiolabeled Antibodies; Pharmacokinetics of Antibodies andTheir Radiolabels; Experimental Radioimmunotherapy and Methods toIncrease Therapeutic Efficacy,” CRC Press, Boca Raton, Chapter 10, pp.115-140, (1995); Fischman, et al. “A Ticket to Ride: PeptideRadiopharmaceuticals,” The Journal of Nuclear Medicine, vol. 34, No. 12,(December 1993). These references are hereby incorporated by referencein their entirety.

In recent years, it has been learned that some cancer cells containgastrin releasing peptide (GRP) receptors (GRP-R) of which there are anumber of subtypes. In particular, it has been shown that several typesof cancer cells have over-expressed or uniquely expressed GRP receptors.For this reason, much research and study have been done on GRP and GRPanalogues which bind to the GRP receptor family. One such analogue isbombesin (BBN), a 14 amino acid peptide (i.e., tetradecapeptide)isolated from frog skin which is an analogue of human GRP and whichbinds to GRP receptors with high specificity and with an affinitysimilar to GRP.

Bombesin and GRP analogues may take the form of agonists or antagonists.Binding of GRP or BBN agonists to the GRP receptor increases the rate ofcell division of these cancer cells and such agonists are internalizedby the cell, while binding of GRP or BBN antagonists generally does notresult in either internalization by the cell or increased rates of celldivision. Such antagonists are designed to competitively inhibitendogenous GRP binding to GRP receptors and reduce the rate of cancercell proliferation. See, e.g., Hoffken, K.; Peptides in Oncology II,Somatostatin Analogues and Bombesin Antagonists (1993), pp. 87-112. Forthis reason, a great deal of work has been, and is being pursued todevelop BBN or GRP analogues that are antagonists. E.g., Davis et al.,Metabolic Stability and Tumor Inhibition of Bombesin/GRP ReceptorAntagonists, Peptides, vol. 13, pp. 401-407, 1992.

In designing an effective compound for use as a diagnostic ortherapeutic agent for cancer, it is important that the drug haveappropriate in vivo targeting and pharmacokinetic properties. Forexample, it is preferable that for a radiopharmaceutical, theradiolabeled peptide have high specific uptake by the cancer cells(e.g., via GRP receptors). In addition, it is also preferred that oncethe radionuclide localizes at a cancer site, it remains there for adesired amount of time to deliver a highly localized radiation dose tothe site.

Moreover, developing radiolabeled peptides that are cleared efficientlyfrom normal tissues is also an important factor for radiopharmaceuticalagents. When biomolecules (e.g., MAb, F_(ab) or peptides) labeled withmetallic radionuclides (via a chelate conjugation), are administered toan animal such as a human, a large percentage of the metallicradionuclide (in some chemical form) can become “trapped” in either thekidney or liver parenchyma (i.e., is not excreted into the urine orbile). Duncan et al.; Indium-111-DiethylenetriaminepentaaceticAcid-Octreotide Is Delivered in Vivo to Pancreatic, Tumor Cell, Renal,and Hepatocyte Lysosomes, Cancer Research 57, pp. 659-671, (Feb. 15,1997). For the smaller radiolabeled biomolecules (i.e., peptides orF_(ab)), the major route of clearance of activity is through the kidneyswhich can also retain high levels of the radioactive metal (i.e.,normally >10-15% of the injected dose). Retention of metal radionuclidesin the kidney or liver is clearly undesirable. Conversely, clearance ofthe radiopharmaceutical from the blood stream too quickly by the kidneyis also undesirable if longer diagnostic imaging or high tumor uptakefor radiotherapy is needed.

Subsequent work, such as that in U.S. Pat. No. 6,200,546 and US2002/0054855 to Hoffman, et. al, hereby incorporated by reference intheir entirety, have attempted to overcome this problem by forming acompound having the general formula X—Y—B wherein X is a group capableof complexing a metal, Y is a covalent bond on a spacer group and B is abombesin agonist binding moiety. Such compounds were reported to havehigh binding affinities to GRP receptors, and the radioactivity wasretained inside of the cells for extended time periods. In addition, invivo studies in normal mice have shown that retention of the radioactivemetal in the kidneys was lower than that known in the art, with themajority of the radioactivity excreted into the urine.

New and improved radiopharmaceutical and other diagnostic compoundswhich have improved pharmacokinetics and improved kidney excretion(i.e., lower retention of the radioactive metal in the kidney) have nowbeen found for diagnostic imaging and therapeutic uses. For diagnosticimaging, rapid renal excretion and low retained levels of radioactivityare critical for improved images. For radiotherapeutic use, slower bloodclearance to allow for higher tumor uptake and better tumor targetingwith low kidney retention are critical.

SUMMARY OF THE INVENTION

In an embodiment of the present invention, there is provided new andimproved compounds for use in diagnostic imaging or radiotherapy. Thecompounds include a chemical moiety capable of complexing a medicallyuseful metal ion or radionuclide (metal chelator) attached to a GRPreceptor targeting peptide by a linker or spacer group. In anotherembodiment, these compounds include an optical label (e.g. a photolabelor other label detectable by light imaging, optoacoustical imaging orphotoluminescence) attached to a GRP receptor targeting peptide by alinker or spacer group.

In general, compounds of the present invention may have the formula:M—N—O—P—Gwherein M is the metal chelator (in the form complexed with a metalradionuclide or not), or the optical label, N—O—P is the linker, and Gis the GRP receptor targeting peptide.

The metal chelator M may be any of the metal chelators known in the artfor complexing with a medically useful metal ion or radionuclide.Preferred chelators include DTPA, DOTA, DO3A, HP-DO3A, EDTA, TETA, EHPG,HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM, or peptidechelators, such as, for example, those discussed herein. The metalchelator may or may not be complexed with a metal radionuclide, and mayinclude an optional spacer such as a single amino acid. Preferred metalradionuclides for scintigraphy or radiotherapy include ^(99m)Tc, ⁵¹Cr,⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y,⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re,¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ²²⁵Ac, ¹⁰⁵Rh, ¹⁰⁹Pd,^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. The choice of metalwill be determined based on the desired therapeutic or diagnosticapplication. For example, for diagnostic purposes the preferredradionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, and ¹¹¹In, with^(99m)Tc, and ¹¹¹In being particularly preferred. For therapeuticpurposes, the preferred radionuclides include ⁶⁴CU, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In,^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu,^(186/188)Re, and ¹⁹⁹Au, with ¹⁷⁷Lu and ⁹⁰Y being particularlypreferred. A most preferred chelator used in compounds of the inventionis 1-substituted 4,7,10-tricarboxymethyl 1,4,7,10 tetraazacyclododecanetriacetic acid (DO3A).

The optical label M may be any of various optical labels known in theart. Preferred labels include, without limitation, optical dyes,including organic chromophores or fluorophores, such as cyanine dyeslight absorbing compounds, light reflecting and scattering compounds,and bioluminescent molecules.

In one embodiment, the linker N—O—P contains at least one non-alphaamino acid.

In another embodiment, the linker N—O—P contains at least onesubstituted bile acid.

In yet another embodiment, the linker N—O—P contains at least onenon-alpha amino acid with a cyclic group.

The GRP receptor targeting peptide may be GRP, bombesin or anyderivatives or analogues thereof. In a preferred embodiment, the GRPreceptor targeting peptide is a GRP or bombesin analogue which acts asan agonist. In a particularly preferred embodiment, the GRP receptortargeting peptide is a bombesin agonist binding moiety disclosed in U.S.Pat. No. 6,200,546 and US 2002/0054855, incorporated herein byreference.

There is also provided a novel method of imaging using the compounds ofthe present invention.

A single or multi-vial kit that contains all of the components needed toprepare the diagnostic or therapeutic agents of the invention isprovided in an exemplary embodiment of the present invention.

There is further provided a novel method for preparing a diagnosticimaging agent comprising the step of adding to an injectable imagingmedium a substance containing the compounds of the present invention.

A novel method of radiotherapy using the compounds of the invention isalso provided, as is a novel method for preparing a radiotherapeuticagent comprising the step of adding to an injectable therapeutic mediuma substance comprising a compound of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphical representation of a series of chemical reactionsfor the synthesis of intermediate C((3β,5β)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid), from A(Methyl-(3β,5β)-3-aminocholan-24-ate) and B((3β,5β)-3-aminocholan-24-oic acid), as described in Example I.

FIG. 1B is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L62), as described in Example I.

FIG. 2A is a graphical representation of the sequential reaction for thesynthesis ofN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L70), as described in Example II.

FIG. 2B is a general graphical representation of the sequential reactionfor the synthesis ofN-[4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L73),N-[3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L115), andN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L116), as described in Example II.

FIG. 2C is a chemical structure of the linker used in the synthesisreaction of FIG. 2B for synthesis ofN-[4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L73), as described in Example II.

FIG. 2D is a chemical structure of the linker used in the synthesisreaction of FIG. 2B for synthesis ofN-[3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L115), as described in Example II.

FIG. 2E is a chemical structure of the linker used in the synthesisreaction of FIG. 2B for synthesis ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L116), as described in Example II.

FIG. 2F is a graphical representation of the sequential reaction for thesynthesis ofN-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]glycyl-4-piperidinecarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L74), as described in Example II.

FIG. 3A is a graphical representation of a series of chemical reactionsfor the synthesis of intermediate(3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxocholan-24-oicacid (C), as described in Example III.

FIG. 3B is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L67), as described in Example III.

FIG. 3C is a chemical structure of (3β,5β)-3-Amino-12-oxocholan-24-oicacid (B), as described in Example III.

FIG. 3D is a chemical structure of(3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxocholan-24-oicacid (C), as described in Example III.

FIG. 3E is a chemical structure ofN-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L67), as described in Example III.

FIG. 4A is a graphical representation of a sequence of reactions toobtain intermediates(3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oicacid (3a) and(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid (3b), as described in Example IV.

FIG. 4B is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L63), as described in Example IV.

FIG. 4C is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β,7α,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L64), as described in Example IV.

FIG. 4D is a chemical structure of(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid (2b), asdescribed in Example IV.

FIG. 4E is a chemical structure of(3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oicacid (3a), as described in Example IV;

FIG. 4F is a chemical structure of(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid (3b), as described in Example IV.

FIG. 4G is a chemical structure ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L63), as described in Example IV.

FIG. 4H is a chemical structure ofN-[(3β,5β,7α,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L64), as described in Example IV.

FIG. 5A is a general graphical representation of the sequential reactionfor the synthesis of4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L71); andTrans-4-[[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]cyclohexylcarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L72) as described in Example V, wherein the linker is from FIG. 5B andFIG. 5C, respectively.

FIG. 5B is a chemical structure of the linker used in compound L71 asshown in FIG. 5A and as described in Example V.

FIG. 5C is a chemical structure of the linker used in compound L72 asshown in FIG. 5A and as described in Example V.

FIG. 5D is a chemical structure of Rink amide resin functionalised withbombesin[7-14] (B), as described in Example V.

FIG. 5E is a chemical structure ofTrans-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]cyclohexanecarboxylicacid (D), as described in Example V;

FIG. 6A is a graphical representation of a sequence of reactions for thesynthesis of intermediate linker2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid (E), asdescribed in Example VI.

FIG. 6B is a graphical representation of a sequence of reactions for thesynthesis of intermediate linker4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acid(H), as described in Example VI.

FIG. 6C is a graphical representation of the synthesis ofN-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L75), as described in Example VI.

FIG. 6D is a graphical representation of the synthesis ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L76), as described in Example VI.

FIG. 7A is a graphical representation of a sequence of reactions for thesynthesis of intermediate linker[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid(E), as described in Example VII.

FIG. 7B is a graphical representation of the synthesis ofN-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L124), as described in Example VII.

FIG. 7C is a chemical structure ofN-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L124), as described in Example VII.

FIG. 8A is a graphical representation of a sequence of reactions for thesynthesis of intermediate4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acid(E), as described in Example VIII.

FIG. 8B is a graphical representation of the synthesis ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L125), as described in Example VIII.

FIG. 8C is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L125), as described in Example VIII.

FIG. 9A is a graphical representation of a reaction for the synthesis of3-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminobenzoic acid,(B), as described in Example IX.

FIG. 9B is a graphical representation of a reaction for the synthesis of6-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminonaphthoic acid(C), as described in Example IX.

FIG. 9C is a graphical representation of a reaction for the synthesis of4-[[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]methylamino]benzoicacid, (D), as described in Example IX.

FIG. 9D is a graphical representation of a reaction for the synthesis ofN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L146);N-[3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L233);N-[6-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]naphthoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L234), andN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]methylamino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L235), as described in Example IX.

FIG. 10A is a graphical representation of a reaction for the synthesisof7-[[Bis(1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triaceticacid 4,10-bis(1,1-dimethylethyl) ester H, as described in Example X.

FIG. 10B is a graphical representation of a reaction for the synthesisofN-[4-[[[[[4,10-Bis(carboxymethyl)-7-(dihydroxyphosphinyl)methyl-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucil-L-methioninamide,(L237), as described in Example X.

FIG. 11A is a graphical representation of a reaction for the synthesisofN,N-Dimethylglycyl-L-serinyl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L238), as described in Example XI.

FIG. 11B is a graphical representation of a reaction for the synthesisofN,N-Dimethylglycyl-L-serinyl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-(3β,5β,7α,12α)-3-amino-7,12-dihydroxy-24-oxocholan-24-yl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L239), as described in Example XI.

FIG. 12A is a graphical representation of a reaction for the synthesisof4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methoxybenzoicacid (A), as described in Example XII.

FIG. 12B is a graphical representation of a reaction for the synthesisof4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoicacid, (D), as described in Example XII.

FIG. 12C is a graphical representation of a reaction for the synthesisof4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methylbenzoicacid (E), as described in Example XII.

FIG. 12D is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]glycyl]amino]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-1-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L240) as described in Example XII.

FIG. 12E is a chemical structure of compoundN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododec-1-yl]acetyl]glycyl]amino]3-chlorobenzoyl]L-glutaminyl-L-tryptophyl-1-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L241) as described in Example XII.

FIG. 12F is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododec-1-yl]acetyl]glycyl]amino]3-methylbenzoyl]L-glutaminyl-L-tryptophyl-1-alanyl-L-valyl-glycyl-L-histidyl-leucyl-L-methioninamide(L242), as described in Example XII.

FIG. 13A is a graphical representation of a reaction for the synthesisof4-[N,N′-Bis[2-[(9-H-fluoren-9-ylmethoxy)carbonyl]aminoethyl]amino]-4-oxobutanoicacid, (D), as described in Example XIII.

FIG. 13B is a graphical representation of a reaction for the synthesisofN-[4-[[4-[Bis[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethyl]amino-1,4-dioxobutyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L244), as described in Example XIII.

FIG. 13C is a chemical structure of compound L244, as described inExample XIII.

FIG. 14A and FIG. 14B are graphical representations of the binding andcompetition curves described in Example XLIII.

FIG. 15A is a graphical representation of the results of radiotherapyexperiments described in Example LV.

FIG. 15B is a graphical representation of the results of otherradiotherapy experiments described in Example LV.

FIG. 16 is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododec-1-yl]acetyl]glycyl]amino]-L-Lysinyl-(3,6,9)-trioxaundecane-1,11-dicarboxylicacid-3,7-dideoxy-3-aminocholicacid)-L-arginyl-L-glutaminyl-L-triptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L65).

FIG. 17 is a chemical structure ofN-[2-S-[[[[[12α-Hydroxy-17α-(1-methyl-3-carboxypropyl)etiocholan-3β-carbamoylmethoxyethoxyethoxyacetyl]-amino-6-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]hexanoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L66).

FIG. 18A is a chemical structure ofN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L70).

FIG. 18B is a chemical structureN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]-3-carboxypropionyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L114).

FIG. 18C is a chemical structureN-[4-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]-2-hydroxy-3-propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L144).

FIG. 18D is a chemical structureN-[(3β,5β,7α,12α)-3-[[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxyethoxy]acetyl]amino]-7,12-dihydroxycholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamine(L69).

FIG. 18E is a chemical structure ofN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L146).

FIG. 19 discloses chemical structures of intermediates which may be usedto prepare compounds L64 and L70 as described in Example LVI.

FIG. 20 is a graphical representation of the preparation of L64 usingsegment coupling as described in Example LVI.

FIG. 21 is a graphical representation of the preparation of(1R)-1-(Bis{2-[bis(carboxymethyl)amino]ethyl}amino)propane-3-carboxylicacid-1-carboxyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L201).

FIG. 22A is a graphical representation of chemical structure of chemicalintermediates used to prepare L202.

FIG. 22B is a graphical representation of the preparation ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-4-hydrazinobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L202).

FIG. 23A is a graphical representation of chemical structure of chemicalintermediates used to prepare L203.

FIG. 23B is a graphical representation of the preparation ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L203).

FIG. 24 is a graphical representation of the preparation ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L204).

FIG. 25 is a graphical representation of the preparation ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L205).

FIG. 26A is a graphical representation of chemical structures ofchemical intermediates used to prepare L206.

FIG. 26B is a graphical representation of the preparation ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-[4′-Amino-2′-methylbiphenyl-4-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L206).

FIG. 27A is a graphical representation of chemical structures ofchemical intermediates used to prepare L207.

FIG. 27B is a graphical representation of the preparation ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-[3′-amino-biphenyl-3-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L207).

FIG. 28 is a graphical representation of the preparation ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-[1,2-d]aminoethyl-terephthalyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L208).

FIG. 29A is a graphical representation of chemical structures ofchemical intermediates used to prepare L209.

FIG. 29B is a graphical representation of the preparation of L209.

FIG. 30A is a graphical representation of chemical structures ofchemical intermediates used to prepare L210.

FIG. 30B is a chemical structure of L210.

FIG. 31 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL211.

FIG. 32 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutamyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL212.

FIG. 33 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninecarboxylate L213.

FIG. 34 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL214.

FIG. 35 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-arginyl-L-leucyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL215.

FIG. 36 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-arginyl-L-tyrosinyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL216.

FIG. 37 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-lysyl-L-tyrosinyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL217.

FIG. 38 is a chemical structure of L218.

FIG. 39 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-aminopentyl,L219.

FIG. 40 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-serinyl-L-valyl-D-alanyl-L-histidyl-L-leucyl-L-methioninamide,L220.

FIG. 41 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-leucinamide,L221.

FIG. 42 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-tyrosinyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide,L222.

FIG. 43 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide,L223.

FIG. 44 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-glycyl-L-histidyl-L-phenylalanyl-L-leucinamide,L224.

FIG. 45 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-valinyl-glycyl-L-serinyl-L-phenylalanyl-L-methioninamide,L225.

FIG. 46 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-histidyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L226.

FIG. 47 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-serinyl-L-phenylalanyl-L-methioninamideL227.

FIG. 48 is a chemical structure ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-phenylalanyl-L-methioninamide,L228.

FIG. 49A is a graphical representation of a reaction for the synthesisof(3β,5β,7α,12α)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oicacid (B) as described in Example LVII.

FIG. 49B is a graphical representation of a reaction for the synthesisofN-[3β,5β,7α,12α)-3-[[[2-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L69), as described in Example LVII.

FIG. 50 is a graphical representation of a reaction for the synthesis ofN-[4-[2-Hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L144), as described in Example LVIII.

FIG. 51 is a chemical structure of L300.

FIG. 52 is a TOCSY spectrum of Lu-L70 in DMSO-d₆ at 25° C.

FIG. 53 is a COSY spectrum of Lu-L70 in DMSO-d₆ at 25° C.

FIG. 54 is a NOESY spectrum of Lu-L70 in DMSO-d₆ at 25° C.

FIG. 55 is a gHSQC spectrum of Lu-L70 in DMSO-d₆ at 25° C.

FIG. 56 is a gHMBC spectrum of Lu-L70 in DMSO-d₆ at 25° C.

FIG. 57 is a gHSQCTOCSY spectrum of Lu-L70 in DMSO-d₆ at 25° C.

FIG. 58 is a Regular 1H-NMR (bottom) and selective homo-decoupling ofthe water peak at 3.5 ppm of Lu-L70 in DMSO-d₆ at 15° C.

FIG. 59 is a TOCSY Spectrum of ¹⁷⁵Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂(SEQ ID NO: 1) in DMSO-d₆ at 25° C.

FIG. 60 is a chemical structure of L70.

FIG. 61 is a chemical structure of ¹⁷⁵Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂(SEQ ID NO: 1)

FIG. 62 is a chemical structure of ¹⁷⁵Lu-L70 with a bound watermolecule.

FIG. 63 is a chemical structure of L301.

Abbreviations Used In the Application Aoc- 8-aminooctanoic acid Apa3-3-aminopropionic acid Abu4- 4-aminobutanoic acid Adca3- (3β,5β7α,12α)-3-amino-7,12-dihydroxycholan-24- oic acid or3-Amino-3-deoxycholic acid Ah12ca-(3β,5β,12α)-3-amino-12-hydroxycholan-24-oic acid Akca-(3β,5β,7α,12α)-3-amino-12-oxacholan-24-oic acid Cha- L-CyclohexylalanineNa11- L-1-Naphthylalanine Bip- L-Biphenylalanine Mo3abz4-3-Methoxy-4-aminobenzoic acid or 4-aminomethyl-3- methoxybenzoic acidBpa4- 4-benzoylphenylalanine C13abz4- 3-Chloro-4-aminobenzoic acidM3abz4- 3-methyl-4-aminobenzoic acid Ho3abz4- 3-hydroxy-4-aminobenzoicacid Hybz4- 4-hydrazinobenzoic acid Nmabz4- 4-methylaminobenzoic acidMo3amb4- 3-methoxy-4-aminobenzoic acid Amb4- 4-aminomethylbenzoic acidAeb4- 4-(2-aminoethoxy)benzoic acid Dae- 1,2-diaminoethyl Tpa-Terephthalic acid A4m2biphc4- 4′-Amino-2′-methyl biphenyl-4-carboxylicacid A3biphc3- 3-amino-3′-biphenylcarboxylic acid Amc4-trans-4-aminomethylcyclohexane carboxylic acid Aepa4-N-4-aminoethyl-N-1-piperazine-acetic acid Inp- Isonipecotic acid Pia1-N-1-piperazineacetic acid Ckbp-4-(3-Carboxymethyl-2-keto-1-benzimidazoyl)- piperidine Abz33-Aminobenzoic acid Abz4 4-Aminobenzoic acid J 8-amino-3,6-dioxaoctanoicacid Ava5 5-Aminovaleric acid f (D)-Phe y (D)-Tyr Ala2 Beta-alanine(also Bala)

DETAILED DESCRIPTION OF THE INVENTION

In the following description, various aspects of the present inventionwill be further elaborated. For purposes of explanation, specificconfigurations and details are set forth in order to provide a thoroughunderstanding of the present invention. However, it will also beapparent to one skilled in the art that the present invention may bepracticed without the specific details. Furthermore, well known featuresmay be omitted or simplified in order not to obscure the presentinvention.

In an embodiment of the present invention, there are provided new andimproved compounds for use in diagnostic imaging or radiotherapy. Thecompounds include an optical label or a chemical moiety capable ofcomplexing a medically useful metal ion or radionuclide (metal chelator)attached to a GRP receptor targeting peptide by a linker or spacergroup.

In general, compounds of the present invention may have the formula:M—N—O—P—Gwherein M is the metal chelator (in the form complexed with a metalradionuclide or not), or an optical label, N—O—P is the linker, and G isthe GRP receptor targeting peptide. Each of the metal chelator, opticallabel, linker, and GRP receptor targeting peptide is described in thediscussion that follow.

In another embodiment of the present invention, there is provided a newand improved linker or spacer group which is capable of linking anoptical label or a metal chelator to a GRP receptor targeting peptide.In general, linkers of the present invention may have the formula:N—O—Pwherein each of N, O and P are defined throughout the specification.

Compounds meeting the criteria defined herein were discovered to haveimproved pharmacokinetic properties compared to other GRP receptortargeting peptide conjugates known in the art. For example, compoundscontaining the linkers of the present invention were retained in thebloodstream longer, and thus had a longer half life than prior knowncompounds. The longer half life was medically beneficial because itpermitted better tumor targeting which is useful for diagnostic imaging,and especially for therapeutic uses, where the cancerous cells andtumors receive greater amounts of the radiolabeled peptides.Additionally, compounds of the present invention had improved tissuereceptor specificity compared to prior art compounds.

1A. Metal Chelator

The term “metal chelator” refers to a molecule that forms a complex witha metal atom, wherein said complex is stable under physiologicalconditions. That is, the metal will remain complexed to the chelatorbackbone in vivo. More particularly, a metal chelator is a molecule thatcomplexes to a radionuclide metal to form a metal complex that is stableunder physiological conditions and which also has at least one reactivefunctional group for conjugation with the linker N—O—P. The metalchelator M may be any of the metal chelators known in the art forcomplexing a medically useful metal ion or radionuclide. The metalchelator may or may not be complexed with a metal radionuclide.Furthermore, the metal chelator can include an optional spacer such as,for example, a single amino acid (e.g., Gly) which does not complex withthe metal, but which creates a physical separation between the metalchelator and the linker.

The metal chelators of the invention may include, for example, linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelators (see also, U.S.Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556,U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142, the disclosures ofwhich are incorporated by reference in their entirety), and otherchelators known in the art including, but not limited to, HYNIC, DTPA,EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see also U.S.Pat. No. 5,720,934). For example, N₄ chelators are described in U.S.Pat. Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and5,688,487, the disclosures of which are incorporated by reference intheir entirety. Certain N₃S chelators are described in PCT/CA94/00395,PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. Nos. 5,662,885;5,976,495; and 5,780,006, the disclosures of which are incorporated byreference in their entirety. The chelator may also include derivativesof the chelating ligand mercapto-acetyl-glycyl-glycyl-glycine (MAG3),which contains an N₃S, and N₂S₂ systems such as MAMA(monoamidemonoaminedithiols), DADS (N2S diaminedithiols), CODADS and thelike. These ligand systems and a variety of others are described in Liuand Edwards, Chem. Rev. 1999, 99, 2235-2268 and references therein, thedisclosures of which are incorporated by reference in their entirety.

The metal chelator may also include complexes containing ligand atomsthat are not donated to the metal in a tetradentate array. These includethe boronic acid adducts of technetium and rhenium dioximes, such asthose described in U.S. Pat. Nos. 5,183,653; 5,387,409; and 5,118,797,the disclosures of which are incorporated by reference in theirentirety.

Examples of preferred chelators include, but are not limited to,diethylenetriamine pentaacetic acid (DTPA),1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10-tetraazacyclododecanetriacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA),4-carbonylmethyl-10-phosphonomethyl-1,4,7,10-Tetraazacyclododecane-1,7-diaceticacid (Cm4pm10d2a); and1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-Cl-EHPG, 5-Br-EHPG,5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, anddibenzyl-DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid(HBED) and derivatives thereof; the class of macrocyclic compounds whichcontain at least 3 carbon atoms, more preferably at least 6, and atleast two heteroatoms (O and/or N), which macrocyclic compounds canconsist of one ring, or two or three rings joined together at the heteroring elements, e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, whereNOTA is 1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA,benzo-DOTMA, where DOTMA is1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraaceticacid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM).Examples of representative chelators and chelating groups contemplatedby the present invention are described in WO 98/18496, WO 86/06605, WO91/03200, WO 95/28179, WO 96/23526, WO 97/36619, PCT/US98/01473,PCT/US98/20182, and U.S. Pat. No. 4,899,755, U.S. Pat. No. 5,474,756,U.S. Pat. No. 5,846,519 and U.S. Pat. No. 6,143,274, each of which ishereby incorporated by reference in its entirety.

Particularly preferred metal chelators include those of Formula 1, 2 and3 (for ¹¹¹In and radioactive lanthanides, such as, for example ¹⁷⁷Lu,⁹⁰Y, ¹⁵³Sm, and ¹⁶⁶Ho) and those of Formula 4, 5 and 6 (for radioactive^(99m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re) set forth below. These and other metalchelating groups are described in U.S. Pat. Nos. 6,093,382 and5,608,110, which are incorporated by reference in their entirety.Additionally, the chelating group of formula 3 is described in, forexample, U.S. Pat. No. 6,143,274; the chelating group of formula 5 isdescribed in, for example, U.S. Pat. Nos. 5,627,286 and 6,093,382, andthe chelating group of formula 6 is described in, for example, U.S. Pat.Nos. 5,662,885; 5,780,006; and 5,976,495, all of which are incorporatedby reference. Specific metal chelators of formula 6 includeN,N-dimethylGly-Ser-Cys; N,N-dimethylGly-Thr-Cys;N,N-diethylGly-Ser-Cys; N,N-dibenzylGly-Ser-Cys; and other variationsthereof. For example, spacers which do not actually complex with themetal radionuclide such as an extra single amino acid Gly, may beattached to these metal chelators (e.g., N,N-dimethylGly-Ser-Cys-Gly;N,N-dimethylGly-Thr-Cys-Gly; N,N-diethylGly-Ser-Cys-Gly;N,N-dibenzylGly-Ser-Cys-Gly). Other useful metal chelators such as allof those disclosed in U.S. Pat. No. 6,334,996, also incorporated byreference (e.g., Dimethylgly-L-t-Butylgly-L-Cys-Gly;Dimethylgly-D-t-Butylgly-L-Cys-Gly; Dimethylgly-L-t-Butylgly-L-Cys,etc.)

Furthermore, sulfur protecting groups such as Acm (acetamidomethyl),trityl or other known alkyl, aryl, acyl, alkanoyl, aryloyl, mercaptoacyland organothiol groups may be attached to the cysteine amino acid ofthese metal chelators.

Additionally, other useful metal chelators include:

In the above Formulas 1 and 2, R is alkyl, preferably methyl. In theabove Formulas 5a and 5b, X is either CH₂ or O; Y is C₁-C₁₀ branched orunbranched alkyl; aryl, aryloxy, arylamino, arylaminoacyl;arylalkyl—where the alkyl group or groups attached to the aryl group areC₁-C₁₀ branched or unbranched alkyl groups, C₁-C₁₀ branched orunbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl orpolyhydroxy-polyalkoxyalkyl groups; J is optional, but if present isC(═O)—, OC(═O)—, SO₂—, NC(═O)—, NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—,N═N—, homopolyamides or heteropolyamines derived from synthetic ornaturally occurring amino acids; all where n is 1-100. Other variants ofthese structures are described, for example, in U.S. Pat. No. 6,093,382.In Formula 6, the group S—NHCOCH₃ may be replaced with SH or S—Z whereinZ is any of the known sulfur protecting groups such as those describedabove. Formula 7 illustrates one embodiment of t-butyl compounds usefulas a metal chelator. The disclosures of each of the foregoing patents,applications and references are incorporated by reference in theirentirety.

In a preferred embodiment, the metal chelator includes cyclic or acyclicpolyaminocarboxylic acids such as DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA(diethylenetriaminepentaacetic acid), DTPA-bismethylamide,DTPA-bismorpholineamide, Cm4pm10d2a(1,4-carbonylmethyl-10-phosphonomethyl-1,4,7,10-Tetraazacyclododecane-1,7-diaceticacid), DO3AN-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl,HP-DO3A, DO3A-monoamide and derivatives thereof.

Preferred metal radionuclides for scintigraphy or radiotherapy include^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb,¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh,¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au and oxides ornitrides thereof. The choice of metal will be determined based on thedesired therapeutic or diagnostic application. For example, fordiagnostic purposes (e.g., to diagnose and monitor therapeutic progressin primary tumors and metastases), the preferred radionuclides include⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, and ¹¹¹In, with ^(99m)Tc and ¹¹¹In beingespecially preferred. For therapeutic purposes (e.g., to provideradiotherapy for primary tumors and metastasis related to cancers of theprostate, breast, lung, etc.), the preferred radionuclides include ⁶⁴Cu,⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb,¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au, with ¹⁷⁷Lu and ⁹⁰Y being particularlypreferred. ^(99m)Tc is particularly useful and is a preferred fordiagnostic radionuclide because of its low cost, availability, imagingproperties, and high specific activity. The nuclear and radioactiveproperties of ^(99m)Tc make this isotope an ideal scintigraphic imagingagent. This isotope has a single photon energy of 140 keV and aradioactive half-life of about 6 hours, and is readily available from a⁹⁹Mo-^(99m)Tc generator. For example, the ^(99m)Tc labeled peptide canbe used to diagnose and monitor therapeutic progress in primary tumorsand metastases. Peptides labeled with ¹⁷⁷Lu, ⁹⁰Y or other therapeuticradionuclides can be used to provide radiotherapy for primary tumors andmetastasis related to cancers of the prostate, breast, lung, etc.

1B. Optical Labels

In an exemplary embodiment, the compounds of the invention may beconjugated with photolabels, such as optical dyes, including organicchromophores or fluorophores, having extensive delocalized ring systemsand having absorption or emission maxima in the range of 400-1500 nm.The compounds of the invention may alternatively be derivatized with abioluminescent molecule. The preferred range of absorption maxima forphotolabels is between 600 and 1000 nm to minimize interference with thesignal from hemoglobin. Preferably, photoabsorption labels have largemolar absorptivities, e.g. >10⁵ cm⁻¹ M⁻¹, while fluorescent optical dyeswill have high quantum yields. Examples of optical dyes include, but arenot limited to those described in WO 98/18497, WO 98/18496, WO 98/18495,WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO98/47538, and references cited therein. For example, the photolabels maybe covalently linked directly to compounds of the invention, such as,for example, compounds comprised of GRP receptor targeting peptides andlinkers of the invention. Several dyes that absorb and emit light in thevisible and near-infrared region of electromagnetic spectrum arecurrently being used for various biomedical applications due to theirbiocompatibility, high molar absorptivity, and/or high fluorescencequantum yields. The high sensitivity of the optical modality inconjunction with dyes as contrast agents parallels that of nuclearmedicine, and permits visualization of organs and tissues without theundesirable effect of ionizing radiation. Cyanine dyes with intenseabsorption and emission in the near-infrared (NIR) region areparticularly useful because biological tissues are optically transparentin this region. For example, indocyanine green, which absorbs and emitsin the NIR region has been used for monitoring cardiac output, hepaticfunctions, and liver blood flow and its functionalized derivatives havebeen used to conjugate biomolecules for diagnostic purposes (R. B.Mujumdar, L. A. Ernst, S. R. Mujumdar, et al., Cyanine dye labelingreagents: Sulfoindocyanine succinimidyl esters. Bioconjugate Chemistry,1993, 4(2), 105-111; Linda G. Lee and Sam L. Woo. “N-Heteroaromatic ionand iminium ion substituted cyanine dyes for use as fluorescent labels”,U.S. Pat. No. 5,453,505; Eric Hohenschuh, et al. “Light imaging contrastagents”, WO 98/48846; Jonathan Turner, et al. “Optical diagnostic agentsfor the diagnosis of neurodegenerative diseases by means of nearinfra-red radiation”, WO 98/22146; Kai Licha, et al. “In-vivo diagnosticprocess by near infrared radiation”, WO 96/17628; Robert A. Snow, etal., Compounds, WO 98/48838. Various imaging techniques and reagents aredescribed in U.S. Pat. Nos. 6,663,847, 6,656,451, 6,641,798, 6,485,704,6,423,547, 6,395,257, 6,280,703, 6,277,841, 6,264,920, 6,264,919,6,228,344, 6,217,848, 6,190,641, 6,183,726, 6,180,087, 6,180,086,6,180,085, 6,013,243, and published U.S. Patent Applications 2003185756,20031656432, 2003158127, 2003152577, 2003143159, 2003105300, 2003105299,2003072763, 2003036538, 2003031627, 2003017164, 2002169107, 2002164287,and 2002156117. All of the above references are incorporated byreference in their entirety.

2A. Linkers Containing at Least One Non-Alpha Amino Acid

In one embodiment of the invention, the linker N—O—P contains at leastone non-alpha amino acid. Thus, in this embodiment of the linker N—O—P,

-   -   N is 0 (where 0 means it is absent), an alpha or non-alpha amino        acid or other linking group;    -   O is an alpha or non-alpha amino acid; and    -   P is 0, an alpha or non-alpha amino acid or other linking group,    -   wherein at least one of N, O or P is a non-alpha amino acid.        Thus, in one example, N=Gly, O=a non-alpha amino acid, and P=0.

Alpha amino acids are well known in the art, and include naturallyoccurring and synthetic amino acids.

Non-alpha amino acids are also known in the art and include those whichare naturally occurring or synthetic. Preferred non-alpha amino acidsinclude:

-   8-amino-3,6-dioxaoctanoic acid;-   N-4-aminoethyl-N-1-acetic acid; and-   polyethylene glycol derivatives having the formula    NH₂—(CH₂CH₂O)_(n)—CH₂CO₂H or NH₂—(CH₂CH₂O)_(n)—CH₂CH₂CO₂H where n=2    to 100.

Examples of compounds having the formula M—N—O—P—G which contain linkerswith at least one non-alpha amino acid are listed in Table 1. Thesecompounds may be prepared using the methods disclosed herein,particularly in the Examples, as well as by similar methods known to oneskilled in the art.

TABLE 1 Compounds Containing Linkers With At Least One Non-alpha AminoAcid Com- HPLC HPLC pound method¹ RT² MS³ IC50⁵ M N O P G* L1 10-40% B5.43 1616.6 5 N,N- Lys 8-amino- none BBN(7-14) dimethylglycine-Ser- 3,6-Cys(Acm)-Gly dioxaocta- noic acid L2 10-40% B 5.47 1644.7 3 N,N- Arg8-amino- none BBN(7-14) dimethylglycine-Ser- 3,6- Cys(Acm)-Glydioxaocta- noic acid L3 10-40% B 5.97 1604.6 >50 N,N- Asp 8-amino- noneBBN(7-14) dimethylglycine-Ser- 3,6- Cys(Acm)-Gly dioxaocta- noic acid L410-40% B 5.92 1575.5 4 N,N- Ser 8-amino- none BBN(7-14)dimethylglycine-Ser- 3,6- Cys(Acm)-Gly dioxaocta- noic acid L5 10-40% B5.94 1545.5 9 N,N- Gly 8-amino- none BBN(7-14) dimethylglycine-Ser- 3,6-Cys(Acm)-Gly dioxaocta- noic acid L6 10-30% B 7.82 1639 >50 N,N- Glu8-amino- none BBN(7-14) (M + dimethylglycine-Ser- 3,6- Na) Cys(Acm)-Glydioxaocta- noic acid L7 10-30% B 8.47 1581 7 N,N- Dala 8-amino- noneBBN(7-14) (M + dimethylglycine-Ser- 3,6- Na) Cys(Acm)-Gly dioxaocta-noic acid L8 10-30% B 6.72 1639 4 N,N- 8-amino-3,6- Lys none BBN(7-14)(M + dimethylglycine-Ser- dioxaocta- Na) Cys(Acm)-Gly noic acid L910-30% B 7.28 823.3 6 N,N- 8-amino-3,6- Arg none BBN(7-14) (M +dimethylglycine-Ser- dioxaocta- 2/2) Cys(Acm)-Gly noic acid L10 10-30% B7.94 1625.6 >50 N,N- 8-amino-3,6- Asp none BBN(7-14) (M +dimethylglycine-Ser- dioxaocta- Na) Cys(Acm)-Gly noic acid L11 10-30% B7.59 1575.6 36 N,N- 8-amino-3,6- Ser none BBN(7-14) dimethylglycine-Ser-dioxaocta- Cys(Acm)-Gly noic acid L12 10-30% B 7.65 1567.5 >50 N,N-8-amino-3,6- Gly none BBN(7-14) (M + dimethylglycine-Ser- dioxaocta- Na)Cys(Acm)-Gly noic acid L13 10-30% B 7.86 1617.7 >50 N,N- 8-amino-3,6-Glu none BBN(7-14) dimethylglycine-Ser- dioxaocta- Cys(Acm)-Gly noicacid L14 10-30% B 7.9 1581.7 11 N,N- 8-amino-3,6- Dala none BBN(7-14)(M + dimethylglycine-Ser- dioxaocta- Na) Cys(Acm)-Gly noic acid L1510-30% B 7.84 1656.8 11.5 N,N- 8-amino-3,6- 8-amino- none BBN(7-14) (M +dimethylglycine-Ser- dioxaocta- 3,6- Na) Cys(Acm)-Gly noic aciddioxaocta- noic acid L16 10-30% B 6.65 1597.4 17 N,N- 8-amino-3,6- 2,3-none BBN(7-14) (M + dimethylglycine-Ser- dioxaocta- diaminopro- Na)Cys(Acm)Gly noic acid pionic acid L17 10-30% B 7.6 1488.6 8 N,N- none8-amino- none BBN(7-14) dimethylglycine-Ser- 3,6- Cys(Acm)-Glydioxaocta- noic acid L18 10-30% B 7.03 1574.6 7.8 N,N- 2,3- 8-amino-none BBN(7-14) dimethylglycine-Ser- diaminopro- 3,6- Cys(Acm)-Gly pionicacid dioxaocta- noic acid L19 10-35% B 5.13 1603.6 >50 N,N- Asp 8-amino-Gly BBN(7-14) dimethylglycine-Ser- 3,6- Cys(Acm)-Gly dioxaocta- noicacid L20 10-35% B 5.19 1603.6 37 N,N- 8-amino-3,6- Asp Gly BBN(7-14)dimethylglycine-Ser- dioxaocta- Cys(Acm)-Gly noic acid L21 10-35% B 5.041575.7 46 N,N- 8-amino-3,6- Ser Gly BBN(7-14) dimethylglycine-Ser-dioxaocta- Cys(Acm)-Gly noic acid L22 10-35% B 4.37 1644.7 36 N,N-8-amino-3,6- Arg Gly BBN(7-14) dimethylglycine-Ser- dioxaocta-Cys(Acm)-Gly noic acid L23 10-35% B 5.32 1633.7 >50 N,N- 8-amino-3,6-8-amino- Gly BBN(7-14) dimethylglycine-Ser- dioxaocta- 3,6- Cys(Acm)-Glynoic acid dioxaocta- noic acid L24 10-35% B 4.18 1574.6 38 N,N-8-amino-3,6- 2,3- Gly BBN(7-14) dimethylglycine-Ser- dioxaocta-diaminopro- Cys(Acm)-Gly noic acid pionic acid L25 10-35% B 4.24 1616.626 N,N- 8-amino-3,6- Lys Gly BBN(7-14) dimethylglycine-Ser- dioxaocta-Cys(Acm)-Gly noic acid L26 10-35% B 4.45 1574.6 30 N,N- 2,3- 8-amino-Gly BBN(7-14) dimethylglycine-Ser- diaminopro- 3,6- Cys(Acm)-Gly pionicacid dioxaocta- noic acid L27 10-35% B 4.38 1627.3 >50 N,N- N-4- Aspnone BBN(7-14) dimethylglycine-Ser- aminoethyl- Cys(Acm)-Gly N-1-piperazine- acetic acid L28 10-35% B 4.1 1600.3 25 N,N- N-4- Ser noneBBN(7-14) dimethylglycine-Ser- aminoethyl- Cys(Acm)-Gly N-1- piperazine-acetic acid L29 10-35% B 3.71 1669.4 36 N,N- N-4- Arg none BBN(7-14)dimethylglycine-Ser- aminoethyl- Cys(Acm)-Gly N-1- piperazine- aceticacid L30 10-35% B 4.57 1657.2 36 N,N- N-4- 8-amino- none BBN(7-14)dimethylglycine-Ser- aminoethyl- 3,6- Cys(Acm)-Gly N-1- dioxaocta-piperazine- noic acid acetic acid L31 10-35% B 3.69 1598.3 >50 N,N- N-4-2,3- none BBN(7-14) dimethylglycine-Ser- aminoethyl- diaminopro-Cys(Acm)-Gly N-1- pionic acid piperazine- acetic acid L32 10-35% B 3.511640.3 34 N,N- N-4- Lys none BBN(7-14) dimethylglycine-Ser- aminoethyl-Cys(Acm)-Gly N-1- piperazine- acetic acid L33 10-35% B 4.29 1584.5 >50N,N- N-1- Asp none BBN(7-14) dimethylglycine-Ser- piperazine-Cys(Acm)-Gly acetic acid L34 10-35% B 4.07 1578.7 38 N,N- N-1- Ser noneBBN(7-14) (M + dimethylglycine-Ser- piperazine- Na) Cys(Acm)-Gly aceticacid L35 10-35% B 3.65 1625.6 26 N,N- N-1- Arg none BBN(7-14)dimethylglycine-Ser- piperazine- Cys(Acm)-Gly acetic acid L36 10-35% B4.43 1636.6 7 N,N- N-1- 8-amino- none BBN(7-14) dimethylglycine-Ser-piperazine- 3,6- Cys(Acm)-Gly acetic acid dioxaocta- noic acid L3710-35% B 3.66 1555.7 23 N,N- N-1- 2,3- none BBN(7-14)dimethylglycine-Ser- piperazine- diaminopro- Cys(Acm)-Gly acetic acidpionic acid L38 10-35% B 3.44 1619.6 7 N,N- N-1- Lys none BBN(7-14)dimethylglycine-Ser- piperazine- Cys(Acm)-Gly acetic acid L42 30-50% B5.65 1601.6 25 N,N- 4- 8-amino- none BBN(7-14) dimethylglycine-Ser-Hydroxypro- 3,6- Cys(Acm)-Gly line dioxaocta- noic acid L48 30-50% B4.47 1600.5 40 N,N- 4- 8-amino- none BBN(7-14) dimethylglycine-Ser-aminoproline 3,6- Cys(Acm)-Gly dioxaocta- noic acid L51 15-35% B 5.141673.7 49 N,N- Lys 8-amino- Gly BBN(7-14) dimethylglycine-Ser- 3,6-Cys(Acm)-Gly dioxaocta- noic acid L52 15-35% B 6.08 1701.6 14 N,N- Arg8-amino- Gly BBN(7-14) dimethylglycine-Ser- 3,6- Cys(Acm)-Gly dioxaocta-noic acid L53 15-35% B 4.16 1632.6 10 N,N- Ser 8-amino- Gly BBN(7-14)dimethylglycine-Ser- 3,6- Cys(Acm)-Gly dioxaocta- noic acid L54 15-35% B4.88 1661.6 >50 N,N- Asp 8-amino- Gly BBN(7-14) dimethylglycine-Ser-3,6- Cys(Acm)-Gly dioxaocta- noic acid L55 15-35% B 4.83 1683.4 43 N,N-8-amino-3,6- Asp Gly BBN(7-14) (M + dimethylglycine-Ser- dioxaocta- Na)Cys(Acm)-Gly noic acid L56 15-35% B 4.65 1655.7 4 N,N- 8-amino-3,6- SerGly BBN(7-14) (M + dimethylglycine-Ser- dioxaocta- Na) Cys(Acm)-Gly noicacid L57 15-35% B 4.9 1701.8 50 N,N- 8-amino-3,6- Arg Gly BBN(7-14)dimethylglycine-Ser- dioxaocta- Cys(Acm)-Gly noic acid L58 15-35% B 4.22846.4 >50 N,N- 8-amino-3,6- 8-amino- Gly BBN(7-14) (M +dimethylglycine-Ser- dioxaocta- 3,6- H/2) Cys(Acm)-Gly noic aciddioxaocta- noic acid L59 15-35% B 4.03 1635.5 42 N,N- 8-amino-3,6- 2,3-Gly BBN(7-14) dimethylglycine-Ser- dioxaocta- diaminopro- Cys(Acm)-Glynoic acid pionic acid L60 15-35% B 4.11 1696.6 20 N,N- 8-amino-3,6- LysGly BBN(7-14) (M + dimethylglycine-Ser- dioxaocta- Na) Cys(Acm)-Gly noicacid L61 15-35% B 4.32 1631.4 43 N,N- 2,3- 8-amino- Gly BBN(7-14)dimethylglycine-Ser- diaminopro- 3,6- Cys(Acm)-Gly pionic aciddioxaocta- noic acid L78 20-40% B 6.13 1691.4 35 DO3A-monoamide8-amino-3,6- Diaminopro- none BBN(7-14) (M + dioxaocta- picnic acid Na)noic acid L79 20-40% B 7.72 1716.8 42 DO3A-monoamide 8-amino-3,6-Biphenylala- none BBN(7-14) (M + dioxaocta- nine Na) noic acid L8020-40% B 7.78 1695.9 >50 DO3A-monoamide 8-amino-3,6- Diphenylala- noneBBN(7-14) dioxaocta- nine noic acid L81 20-40% B 7.57 1513.6 37.5DO3A-monoamide 8-amino-3,6- 4- none BBN(7-14) dioxaocta- Benzoylphen-noic acid ylalanine L92 15-30% B 5.63 1571.6 5 DO3A-monoamide 5-8-amino- none BBN(7-14) aminopenta- 3,6- noic acid dioxaocta- noic acidL94 20-36% B 4.19 1640.8 6.2 DO3A-monoamide 8-amino-3,6- D- noneBBN(7-14) (M + dioxaocta- Phenylala- Na) noic acid nine L110 15-45% B5.06 1612.7 36 DO3A-monoamide 8- 8-amino- none BBN(7-14) aminoocta- 3,6-noic acid dioxaocta- noic acid L209 20-40% B 4.62 3072.54 37DO3A-monoamide E(G8-amino- 8- 8- BBN(7-14) over 6 3,6- aminoocta- amino-minutes dioxaocta- noic acid octanoic noic acid-8- acid amino-3,6-dioxaoctanoic acid QWAVGHLM- NH₂) (SEQ ID NO: 1) L210 20-50% B 6.183056.76 11 DO3A-monoamide E(G-Aoa- 8- 8- BBN(7-14) over 10 Aoa-aminoocta- amino- minutes QWAVGHLM- noic acid octanoic NH₂) acid (SEQ IDNO: 1) *BBN(7-14) is [SEQ ID NO: 1] ¹HPLC method refers to the 10 minutetime for the HPLC gradient. ²HPLC RT refers to the retention time of thecompound in the HPLC. ³MS refers to mass spectra where molecular weightis calculated from mass/unit charge (m/e). ⁴IC₅₀ refers to theconcentration of compound to inhibit 50% binding of iodinated bombesinto a GRP receptor on cells.

2B. Linkers Containing at Least One Substituted Bile Acid

In another embodiment of the present invention, the linker N—O—Pcontains at least one substituted bile acid. Thus, in this embodiment ofthe linker N—O—P,

-   -   N is 0 (where 0 means it is absent), an alpha amino acid, a        substituted bile acid or other linking group;    -   O is an alpha amino acid or a substituted bile acid; and    -   P is 0, an alpha amino acid, a substituted bile acid or other        linking group, wherein at least one of N, O or P is a        substituted acid.

Bile acids are found in bile (a secretion of the liver) and are steroidshaving a hydroxyl group and a five carbon atom side chain terminating ina carboxyl group. In substituted bile acids, at least one atom such as ahydrogen atom of the bile acid is substituted with another atom,molecule or chemical group. For example, substituted bile acids includethose having a 3-amino, 24-carboxyl function optionally substituted atpositions 7 and 12 with hydrogen, hydroxyl or keto functionality.

Other useful substituted bile acids in the present invention includesubstituted cholic acids and derivatives thereof. Specific substitutedcholic acid derivatives include:

-   (3β,5β)-3-aminocholan-24-oic acid;-   (3β,5β,12α)-3-amino-12-hydroxycholan-24-oic acid;-   (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid;-   Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholic    acid);-   (3β,5β,7α)-3-amino-7-hydroxy-12-oxocholan-24-oic acid; and-   (3β,5β,7α)-3-amino-7-hydroxycholan-24-oic acid.

Examples of compounds having the formula M—N—O—P—G which contain linkerswith at least one substituted bile acid are listed in Table 2. Thesecompounds may be prepared using the methods disclosed herein,particularly in the Examples, as well as by similar methods known to oneskilled in the art.

TABLE 2 Compounds Containing Linkers With At Least One Substituted BileAcid Com- HPLC HPLC pound method¹ RT² MS³ IC50⁵ M N O P G* L62 20-80% B3.79 1741.2 >50 DO3A-monoamide Gly (3β,5β)-3- none BBN(7-14)aminocholan-24- oic acid L63 20-80% B 3.47 1757.0 23 DO3A-monoamide Gly(3β,5β,12α)-3- none BBN(7-14) amino-12- hydroxycholan- 24-oic acid L6420-50% B 5.31 1773.7 8.5 DO3A-monoamide Gly (3β,5β,7α,12α)- noneBBN(7-14) 3-amino-7,12- dihydroxycholan- 24-oic acid L65 20-80% B 3.572246.2 >50 DO3A-monoamide Gly Lys-(3,6,9- Arg BBN(7-14) trioxaundecane-1,11-dicarbonyl- 3,7- dideoxy-3- aminocholic acid) L66 20-80% 3.792245.8 >50 DO3A-monoamide Gly Lys- Arg BBN(7-14) (3β,5β,7α,12α)-3-amino-7,12- dihydroxycholan- 24-oic acid- 3,6,9- trioxaundecane-1,11-dicarbonyl L67 20-80% 3.25 1756.9 4.5 DO3A-monoamide Gly(3β,5β,7α,12α)- none BBN(7-14) 3-amino-12- oxacholan-24- oic acid L6920-80% 3.25 1861.27 8 DO3A-monoamide 1- (3β,5β,7α,12α)- none BBN(7-14)amino- 3-amino-7,12- 3,6- dihydroxycholan- dioxaoct- 24-oic acid anoicacid L280 — — — — DO3A-monoamide Gly 3β,5β 7α,12α)- none Q-W-A-V-a-3-amino-7,12- H-L-M-NH2 dihydroxycholan- (SEQ ID NO: 15) 24-oic acidL281 — — — — DO3A-monoamide Gly 3β,5β 7α,12α)- f Q-W-A-V- 3-amino-7,12-G-H-L-M-NH2 dihydroxycholan- (SEQ ID NO: 1) 24-oic acid L282 — — — —DO3A-monoamide Gly 3β,5β 7α,12α)- f Q-W-A-V- 3-amino-7,12- G-H-L-L-dihydroxycholan- NH2 24-oic acid (SEQ ID NO: 8) L283 — — — —DO3A-monoamide Gly 3β,5β 7α,12α)- f Q-W-A-V- 3-amino-7,12- G-H-L-NH-dihydroxycholan- pentyl 24-oic acid (SEQ ID NO: 6) L284 — — — —DO3A-monoamide Gly 3β,5β 7α,12α)- y QWAVBala- 3-amino-7,12- HFNle-NH₂dihydroxycholan- (SEQ ID NO: 9) 24-oic acid L285 — — — — DO3A-monoamideGly 3β,5β 7α,12α)- f Q-W-A-V- 3-amino-7,12- Bala-H-F- dihydroxycholan-Nle-NH₂ 24-oic acid (SEQ ID NO: 9) L286 — — — — DO3A-monoamide Gly 3β,5β7α,12α)- none QWAVGHFL- 3-amino-7,12- NH₂ dihydroxycholan- (SEQ ID NO11) 24-oic acid L287 — — — — DO3A-monoamide Gly 3β,5β 7α,12α)- noneQWAVGN 3-amino-7,12- MeHis-LM- dihydroxycholan- NH₂ 24-oic acid (SEQ IDNO: 16) L288 — — — — DO3A-monoamide Gly 3β,5β 7α,12α)- none LWAVGSF-3-amino-7,12- M-NH₂ dihydroxycholan- (SEQ ID NO: 12) 24-oic acid L289 —— — — DO3A-monoamide Gly 3β,5β 7α,12α)- none HWAVGHL- 3-amino-7,12-M-NH₂ dihydroxycholan- (SEQ ID NO: 13) 24-oic acid L290 — — — —DO3A-monoamide Gly 3β,5β 7α,12α)- none LWATGH- 3-amino-7,12- F-M-NH₂dihydroxycholan- (SEQ ID NO: 17) 24-oic acid L291 — — — — DO3A-monoamideGly 3β,5β 7α,12α)- none QWAVGH- 3-amino-7,12- FMNH₂ dihydroxycholan-(SEQ ID NO: 14) 24-oic acid L292 — — — — DO3A-monoamide Gly 3β,5β7α,12α)- QRLGN QWAVGHLM- 3-amino-7,12- NH₂ dihydroxycholan- (SEQ IDNO: 1) 24-oic acid L293 — — — — DO3A-monoamide Gly 3β,5β 7α,12α)- QRYGNQWAVGHLM- 3-amino-7,12- NH₂ dihydroxycholan- (SEQ ID NO: 1) 24-oic acidL294 — — — — DO3A-monoamide Gly 3β,5β 7α,12α)- QKYGN QWAVGHLM-3-amino-7,12- NH₂ dihydroxycholan- (SEQ ID NO: 1) 24-oic acid L295 — — —— Pglu-Q-Lys (DO3A- Gly 3β,5β 7α,12α)- LG-N QWAVGHLM- monoamide)3-amino-7,12- NH₂ dihydroxycholan- (SEQ ID NO: 1) 24-oic acid L303 — — —— DO3A-monoamide Gly 3-amino-3- none QRLGNQWAVGHLM- deoxycholic acid NH₂(SEQ ID NO: 3) L304 — — — — DO3A-monoamide Gly 3-amino-3- noneQRYGNQWAVGHLM- deoxycholic acid NH₂ (SEQ ID NO: 4) L305 — — — —DO3A-monoamide Gly 3-amino-3- none QKYGNQWAVGHLM- deoxycholic acid NH₂(SEQ ID NO: 5) L306 — — — — DO3A-monoamide Gly 3-amino-3- none See FIG.38 for deoxycholic acid structure of targeting peptide *BBN(7-14) is[SEQ ID NO: 1] ¹HPLC method refers to the 10 minute time for the HPLCgradient. ²HPLC RT refers to the retention time of the compound in theHPLC. ³MS refers to mass spectra where molecular weight is calculatedfrom mass/unit charge (m/e). ⁴IC₅₀ refers to the concentration ofcompound to inhibit 50% binding of iodinated bombesin to a GRP receptoron cells.

2C. Linkers Containing at Least One Non-Alpha Amino Acid with a CyclicGroup

In yet another embodiment of the present invention, the linker N—O—Pcontains at least one non-alpha amino acid with a cyclic group. Thus, inthis embodiment of the linker N—O—P,

-   -   N is 0 (where 0 means it is absent), an alpha amino acid, a        non-alpha amino acid with a cyclic group or other linking group;    -   O is an alpha amino acid or a non-alpha amino acid with a cyclic        group; and    -   P is 0, an alpha amino acid, a non-alpha amino acid with a        cyclic group, or other linking group,    -   wherein at least one of N, O or P is a non-alpha amino acid with        a cyclic group.

Non-alpha amino acids with a cyclic group include substituted phenyl,biphenyl, cyclohexyl or other amine and carboxyl containing cyclicaliphatic or heterocyclic moieties. Examples of such include:

-   4-aminobenzoic acid (hereinafter referred to as “Abz4 in the    specification”)-   3-aminobenzoic acid-   4-aminomethyl benzoic acid-   8-aminooctanoic acid-   trans-4-aminomethylcyclohexane carboxylic acid-   4-(2-aminoethoxy)benzoic acid-   isonipecotic acid-   2-aminomethylbenzoic acid-   4-amino-3-nitrobenzoic acid-   4-(3-carboxymethyl-2-keto-1-benzimidazolyl-piperidine-   6-(piperazin-1-yl)-4-(3H)-quinazolinone-3-acetic acid-   (2S,5S)-5-amino-1,2,4,5,6,7-hexahydro-azepino[3,21-hi]indole-4-one-2-carboxylic    acid-   (4S,7R)-4-amino-6-aza-5-oxo-9-thiabicyclo[4.3.0]nonane-7-carboxylic    acid-   3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one    N1-piperazineacetic acid-   N-4-aminoethyl-N-1-piperazineacetic acid-   (3S)-3-amino-1-carboxymethylcaprolactam-   (2S,6S,9)-6-amino-2-carboxymethyl-3,8-diazabicyclo-[4,3,0]-nonane-1,4-dione-   3-amino-3-deoxycholic acid-   4-hydroxybenzoic acid-   4-aminophenylacetic acid-   3-hydroxy-4-aminobenzoic acid-   3-methyl-4-aminobenzoic acid-   3-chloro-4-aminobenzoic acid-   3-methoxy-4-aminobenzoic acid-   6-aminonaphthoic acid-   N,N′-Bis(2-aminoethyl)-succinamic acid

Examples of compounds having the formula M—N—O—P—G which contain linkerswith at least one alpha amino acid with a cyclic group are listed inTable 3. These compounds may be prepared using the methods disclosedherein, particularly in the Examples, as well as by similar methodsknown to one skilled in the art.

TABLE 3 Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl,Cycloalkyl Or Heterocyclic) Carboxylates Com- HPLC HPLC pound method¹RT² MS³ IC50⁵ M N O P G* L70 10-40% B 6.15 1502.6 5 DO3A- Gly4-aminobenzoic none BBN(7-14) monoamide acid L71 20-50% 14.14 59.68 7DO3A- none 4-aminomethyl none BBN(7-14) over 30 (M + monoamide benzoicacid minutes Na) L72 20-50% 13.64 65.73 8 DO3A- none trans-4- noneBBN(7-14) over 30 (M + K) monoamide aminomethyl- minutes cyclohexylcarboxylic acid L73 5-35% 7.01 1489.8 5 DO3A- none 4-(2- none BBN(7-14)monoamide aminoethoxy)ben- zoic acid L74 5-35% 6.49 1494.8 7 DO3A- Glyisonipecotic none BBN(7-14) monoamide acid L75 5-35% 6.96 1458.0 23DO3A- none 2- none BBN(7-14) monoamide aminomethylben- zoic acid L765-35% 7.20] 1502.7 4 DO3A- none 4-aminomethyl- none BBN(7-14) monoamide3-nitrobenzoic acid L77 20-40% B 6.17 1691.8 17.5 DO3A- 8-amino-1-Naphthylala- none BBN(7-14) (M + monoamide 3,6- nine Na) dioxaocta-noic acid L82 20-40% B 6.18 1584.6 8 DO3A- none 4-(3- none BBN(7-14)monoamide carboxymethyl- 2-keto-1- benzimidazolyl- piperidine L83 20-40%B 5.66 1597.5 >50 DO3A- none 6-(piperazin-1- none BBN(7-14) monoamideyl)-4-(3H)- quinazolinone- 3-acetic acid L84 20-40% B 6.31 1555.5 >50DO3A- none (2S,5S)-5- none BBN(7-14) monoamide amino- 1,2,4,5,6,7-hexahydro- azepino[3,21- hi]indole- 4-one-2- carboxylic acid L85 20-40%B 5.92 1525.5 >50 DO3A- none (4S,7R)-4- none BBN(7-14) monoamideamino-6-aza-5- oxo-9- thiabicyclo[4.3. 0]nonane-7- carboxylic acid L8620-40% B 6.46 1598.6 >50 DO3A- none N,N- none BBN(7-14) monoamidedimethylglycine L87 20-40% B 5.47 1593.8 >50 DO3A- none 3- noneBBN(7-14) (M + monoamide carboxymethyl- Na) 1-phenyl-1,3,8-triazaspiro[4.5] decan-4-one L88 20-40% B 3.84 1452.7 >50 DO3A- none N1-none BBN(7-14) monoamide piperazineacetic acid L89 20-40% B 5.68 1518.523 DO3A- none N-4- none BBN(7-14) (M + monoamide aminoethyl-N- Na)1-piperazine- acetic acid L90 20-40% B 7.95 1495.4 50 DO3A- none(3S)-3-amino- none BBN(7-14) monoamide 1- carboxymethyl- caprolactam L9120-40% B 3.97 1535.7 >50 DO3A- none (2S,6S,9)-6- none BBN(7-14)monoamide amino-2- carboxymethyl- 3,8- diazabicyclo- [4,3,0]-nonane-1,4-dione L93 15-30% B 7.57 1564.7 5.8 DO3A- 5- trans-4- none BBN(7-14)monoamide aminopent- aminomethylcy- anoic acid clohexane-1- carboxylicacid L95 15-35% B 5.41 1604.6 14 DO3A- trans-4- D- none BBN(7-14)monoamide aminometh- Phenylalanine ylcyclohex- ane-1- carboxylic acidL96 20-36% B 4.75 1612.7 35 DO3A- 4- 8-amino-3,6- none BBN(7-14)monoamide aminometh- dioxaoctanoic ylbenzoic acid acid L97 15-35% B 5.861598.8 4.5 DO3A- 4-benzoyl- trans-4- none BBN(7-14) monoamide (L)-aminomethylcy- phenylala- clohexane-1- nine carboxylic acid L98 15-35% B4.26 1622.7 16 DO3A- trans-4- Arg none BBN(7-14) monoamide aminometh-ylcyclohex- ane-1- carboxylic acid L99 15-35% B 4.1 1594.7 22 DO3A-trans-4- Lys none BBN(7-14) monoamide aminometh- ylcyclohex- ane-1-carboxylic acid L100 15-35% B 4.18 1613.6 10 DO3A- trans-4- Diphenylala-none BBN(7-14) monoamide aminometh- nine ylcyclohex- ane-1- carboxylicacid L101 15-35% B 5.25 1536.7 25 DO3A- trans-4- 1- none BBN(7-14)monoamide aminometh- Naphthylala- ylcyclohex- nine- ane-1- carboxylicacid L102 15-35% B 5.28 1610.8 9.5 DO3A- trans-4- 8-amino-3,6- noneBBN(7-14) monoamide aminometh- dioxaoctanoic ylcyclohex- acid ane-1-carboxylic acid L103 15-35% B 4.75 1552.7 24 DO3A- trans-4- Ser noneBBN(7-14) monoamide aminometh- ylcyclohex- ane-1- carboxylic acid L10415-35% B 3.91 1551.7 32 DO3A- trans-4- 2,3- none BBN(7-14) monoamideaminometh- diaminopropi- ylcyclohex- onic acid ane-1- carboxylic acidL105 20-45% B 7.68 1689.7 3.5 DO3A- trans-4- Biphenyla- none BBN(7-14)monoamide aminometh- lanine ylcyclohex- ane-1- carboxylic acid L10620-45% B 6.97 1662.7 3.8 DO3A- trans-4- (2S,5S)-5- none BBN(7-14)monoamide aminometh- amino- ylcyclohex- 1,2,4,5,6,7- ane-1- hexahydro-carboxylic azepino[3,21- acid hi]indole- 4-one-2- carboxylic acid L10715-35% B 5.79 1604.7 5 DO3A- trans-4- trans-4- none BBN(7-14) monoamideaminometh- aminomethylcy- ylcyclohex- clohexane-1- ane-1- carboxylicacid carboxylic acid L108 15-45% B 6.38 1618.7 10 DO3A- 8-amino-Phenylalanine none BBN(7-14) monoamide 3,6- dioxaocta- noic acid L10915-45% B 6.85 1612.7 6 DO3A- trans-4- Phenylalanine none BBN(7-14)monoamide aminometh- ylcyclohex- ane-1- carboxylic acid L111 20-45% B3.75 1628.6 8 DO3A- 8- trans-4- none BBN(7-14) monoamide aminoocta-aminomethylcy- noic acid clohexane-1- carboxylic acid L112 20-47% B in3.6 1536.5 4.5 DO3A- none 4′- none BBN(7-14) 9 min monoamideaminomethyl- biphenyl-1- carboxylic acid L113 20-47% B in 3.88 1558.6 5DO3A- none 3′- none BBN(7-14) 9 min (M + monoamide aminomethyl- Na)biphenyl-3- carboxylic acid L114 10-40% B 5.47 1582.8 4.5 CMDOTA Gly4-aminobenzoic none BBN(7-14) acid L124 5-35% B 7.04 1489.9 8.0 DO3A-none 4- none BBN(7-14) monoamide aminomethyl- phenoxyacetic acid L1435-35% B 6.85 1516.8 11 DO3A- Gly 4- none BBN(7-14) monoamideaminophenyl- acetic acid L144 5-35% B 6.85 1462.7 9 HPDO3A none4-phenoxy none BBN(7-14) L145 20-80% B 1.58 1459.8 5 DO3A- none 3- noneBBN(7-14) monoamide aminomethylben- zoic acid L146 20-80% B 1.53 1473.79 DO3A- none 4- none BBN(7-14) monoamide aminomethylphen- ylacetic acidL147 20-80% B 1.68 1489.7 3.5 DO3A- none 4-aminomethyl- none BBN(7-14)monoamide 3- methoxybenzoic acid L201 10-46% B 5.77 1563.7 36 Boa***none Gly 4- BBN(7-14) over 12 amino- minutes benzoic acid L202 10-46% B5.68 1517.74 13 DO3A- none Gly 4- BBN(7-14) over 12 monoamide hydra-minutes zinoben- zoyl L203 10-46% B 5.98 1444.69 9 DO3A- none none 4-BBN(7-14) over 12 monoamide amino- minutes benzoic acid L204 10-46% B5.82 1502.73 50 DO3A- none 4-aminobenzoic Gly BBN(7-14) over 12monoamide acid minutes L205 10-46% B 5.36 1503.72 45 DO3A- Gly 6- noneBBN(7-14) over 12 monoamide Aminonicotinic minutes acid L206 10-46% B7.08 1592.85 4.5 DO3A- Gly 4′-Amino-2′- none BBN(7-14) over 12 monoamidemethyl- minutes biphenyl-4- carboxylic acid L207 10-46% B 7.59 1578.832.5 DO3A- Gly 3′- none BBN(7-14) over 12 monoamide Aminobiphenyl-minutes 3-carboxylic acid L208 10-46% B 5.9 1516.75 7.5 DO3A- Gly 1,2-Tereph- BBN(7-14) over 12 monoamide diaminoethyl thalic minutes acidL211 10-46% B 5.76 1560.77 4 DO3A- Gly Gly 4- BBN(7-14) over 12monoamide amino- minutes benzoic acid L212 10-46% B 6.05 1503.71 NT**DO3A- none Gly 4- EWAVGHLM- over 12 monoamide amino- NH₂ minutes benzoic(SEQ ID NO: 2) acid L213 10-46% B 5.93 1503.71 NT** DO3A- Gly4-aminobenzoic none QWAVGHLM- over 12 monoamide acid OH minutes (SEQ IDNO: 1) L214 10-46% B 7.36 1649.91 NT** DO3A- Gly 4-aminobenzoic (D)-BBN(7-14) over 12 monoamide acid Phe minutes L215 10-46% B 5.08 2071.37NT** DO3A- Gly 4-aminobenzoic none QRLGNQWAVGHLM- over 12 monoamide acidNH₂ minutes (SEQ ID NO: 3) L216 10-46% B 4.94 2121.38 NT** DO3A- Gly4-aminobenzoic none QRYGNQWAVGHLM- over 12 monoamide acid NH₂ minutes(SEQ ID NO: 4) L217 10-46% B 4.38 2093.37 NT** DO3A- Gly 4-aminobenzoicnone QKYGNQWAVGHLM- over 12 monoamide acid NH2 minutes (SEQ ID NO: 5)L218 10-46% B 6.13 2154.45 NT** DO3A- Gly 4-aminobenzoic none See FIG.38 for over 12 monoamide acid structure of minutes targeting peptideL219 10-46% B 8.61 1588.84 NT** DO3A- Gly 4-aminobenzoic (D)- QWAVGHL-over 12 monoamide acid Phe NH-Pentyl minutes (SEQ ID NO: 6) L220 10-46%B 5.96 1516.75 NT** DO3A- Gly 4-aminobenzoic none QWSVaHLM- over 12monoamide acid NH₂ minutes (SEQ ID NO: 7) L221 10-46% B 7.96 1631.87NT** DO3A- Gly 4-aminobenzoic (D)- QWAVGHLL- over 12 monoamide acid PheNH₂ minutes (SEQ ID NO: 8) L222 10-46% B 6.61 1695.91 NT** DO3A- Gly4-aminpbenzoic (D)- QWAV- over 12 monoamide acid Tyr Bala-HF- minutesNle-NH₂ (SEQ ID NO: 9) L223 10-46% B 7.48 1679.91 NT** DO3A- Gly4-aminobenzoic Phe QWAV- over 12 monoamide acid Bala-HF- minutes Nle-NH₂(SEQ ID NO: 9) L224 10-46% B 5.40 1419.57 NT** DO3A- Gly 4-aminobenzoicnone QWAGHFL- over 12 monoamide acid NH₂ minutes (SEQ ID NO: 10) L22510-46% B 8.27 1471.71 NT** DO3A- Gly 4-aminobenzoic none LWAVGSFM- over12 monoamide acid NH₂ minutes (SEQ ID NO: 12) L226 10-46% B 5.12 1523.75NT** DO3A- Gly 4-aminobenzoic none HWAVGHLM- over 12 monoamide acid NH₂minutes (SEQ ID NO: 13) L227 10-46% B 6.61 1523.75 NT** DO3A- Gly4-aminobenzoic none LWAVGSFM- over 12 monoamide acid NH₂ minutes (SEQ IDNO: 12) L228 10-46% B 5.77 1511 NT** DO3A- Gly 4-aminobenzoic noneQWAVGHFM- over 12 monoamide acid NH₂ minutes (SEQ ID NO: 14) L233 5-35%B 7.04 1502.71 4.8 DO3A- Gly 3-aminobenzoic none BBN(7-14) over 10 minmonoamide acid L234 20-80% 1.95 1552.76 3 DO3A- Gly 6- none BBN(7-14)over 10 monoamide aminonaphthoic minutes acid L235 20-80% 1.95 1515.72 7DO3A- Gly 4- none BBN(7-14) over 10 monoamide methylamino- minutesbenzoic acid L237 20-80% 1.52 1538.68 5 Cm4pm10d2a Gly 4-aminobenzoicnone BBN(7-14) over 10 acid minutes L238 5-35% B 7.17 1462.70 1.5 N,N-Gly 4-aminobenzoic none BBN(7-14) over 10 min dimethylgly- acidcine-Ser- Cys(Acm)- Gly L239 20-80% 3.36 1733.16 4.5 N,N- Gly 3-amino-3-none BBN(7-14) over 10 dimethylgly- deoxycholic minutes cine-Ser- acidCys(Acm)- Gly L240 20-80% 1.55 1532.73 4 DO3A- Gly 3-methoxy-4- noneBBN(7-14) over 10 monoamide aminobenzoic minutes acid L241 20-80% 1.631535.68 4 DO3A- Gly 3-chloro-4- none BBN(7-14) over 10 monoamideaminobenzoic minutes acid L242 20-80% 1.55 1516.75 5 DO3A- Gly3-methyl-4- none BBN(7-14) over 10 monoamide aminobenzoic minutes acidL243 20-80% 1.57 1518.70 14 DO3A- Gly 3-hydroxy-4- none BBN(7-14) over10 monoamide aminobenzoic minutes acid L244 5-50% over 4.61 1898.16 >50(DO3A- N,N′- none none BBN(7-14) 10 minutes monoamide)₂ Bis(2- aminoeth-yl)- succinamic acid L300 10-46% — — — DO3A- Gly 4-aminobenzoic noneQWAVGHFL- over 10 monoamide acid NH₂ minutes (SEQ ID NO: 11) L301 20-45%7.18 — — DO3A- none 4- L-1- BBN(7-14) over 15 monoamide aminomethylben-Naphth- minutes zoic acid ylala- nine L302 — — — — DO3A- Gly4-aminobenzoic none QWAVGN monoamide acid MeHis-L- M-NH₂ (SEQ ID NO: 16)*BBN(7-14) is [SEQ ID NO: 1] **NT is defined as “not tested.” ***BOA isdefined as(1.R)-1-(Bis{2-[bis(carboxymethyl)amino]ethyl}amino)propane-1,3-dicarboxylicacid. ¹HPLC method refers to the 10 minute time for the HPLC gradient.²HPLC RT refers to the retention time of the compound in the HPLC. ³MSrefers to mass spectra where molecular weight is calculated frommass/unit charge (m/e). ⁴IC₅₀ refers to the concentration of compound toinhibit 50% binding of iodinated bombesin to a GRP receptor on cells.

A subset of compounds containing preferred linkers and various GRPreceptor targeting peptides are set forth in Table 4. These compoundsmay be prepared using the methods disclosed herein, particularly in theExamples, as well as by similar methods known to one skilled in the art.

TABLE 4 Compounds Containing Linkers of the Invention With Various GRP-RTargeting Moities Com- HPLC HPLC pound method¹ RT² MS³ IC50⁵ M N O P G*L214 10-46% B over 7.36 1649.91 NT** DO3A- Gly 4-aminobenzoic (D)-PheBBN(7-14) 12 minutes monoamide acid L215 10-46% B over 5.08 2071.37 NT**DO3A- Gly 4-aminobenzoic none QRLGNQWAVGHLM- 12 minutes monoamide acidNH₂ (SEQ ID NO: 3) L216 10-46% B over 4.94 2121.38 NT** DO3A- Gly4-aminobenzoic none QRYGNQWAVGHLM- 12 minutes monoamide acid NH₂ (SEQ IDNO: 4) L217 10-46% B over 4.38 2093.37 NT** DO3A- Gly 4-aminobenzoicnone QKYGNQWAVGHLM- 12 minutes monoamide acid NH2 (SEQ ID NO: 5) L21810-46% B over 6.13 2154.45 NT** DO3A- Gly 4-aminobenzoic none See FIG.38 for 12 minutes monoamide acid structure of targeting peptide L21910-46% B over 8.61 1588.84 NT** DO3A- Gly 4-aminobenzoic (D)-PheQWAVGHL- 12 minutes monoamide acid NH-Pentyl (SEQ ID NO: 6) L220 10-46%B over 5.96 1516.75 NT** DO3A- Gly 4-aminobenzoic none QWAVaHLM- 12minutes monoamide acid NH₂ (SEQ ID NO: 15) L221 10-46% B over 7.961631.87 NT** DO3A- Gly 4-aminobenzoic (D)-Phe QWAVGHLL- 12 minutesmonoamide acid NH₂ (SEQ ID NO: 8) L222 10-46% B over 6.61 1695.91 NT**DO3A- Gly 4-aminobenzoic (D)-Tyr QWAV- 12 minutes monoamide acidBala-HF- Nle-NH₂ (SEQ ID NO: 9) L223 10-46% B over 7.48 1679.91 NT**DO3A- Gly 4-aminobenzoic Phe QWAV- 12 minutes monoamide acid Bala-HF-Nle-NH₂ (SEQ ID NO: 9) L224 10-46% B over 5.40 1419.57 NT** DO3A- Gly4-aminobenzoic none QWAGHFL- 12 minutes monoamide acid NH₂ (SEQ ID NO:10) L225 10-46% B over 8.27 1471.71 NT** DO3A- Gly 4-aminobenzoic noneLWAVGSFM- 12 minutes monoamide acid NH₂ (SEQ ID NO: 12) L226 10-46% Bover 5.12 1523.75 NT** DO3A- Gly 4-aminobenzoic none HWAVGHLM- 12minutes monoamide acid NH₂ (SEQ ID NO: 13) L227 10-46% B over 6.611523.75 NT** DO3A- Gly 4-aminobenzoic none LWATGHFM- 12 minutesmonoamide acid NH₂ (SEQ ID NO: 17) L228 10-46% B over 5.77 1511 NT**DO3A- Gly 4-aminobenzoic none QWAVGHFM- 12 minutes monoamide acid NH₂(SEQ ID NO: 14) L280 — — — — DO3A- Gly (3β,5β 7a,12a)- none QWAVaHLM-monoamide 3-amino-7,12- NH₂ dihydroxycho- (SEQ ID NO: 15) lan-24-oicacid L281 — — — — DO3A- Gly (3β,5β 7a,12a)- f QWAVGH- monoamide3-amino-7,12- LM-NH₂ dihydroxycho- (SEQ ID NO: 1) lan-24-oic acid L282 —— — — DO3A- Gly (3β,5β 7a,12a)- f QWAVGHLL- monoamide 3-amino-7,12- NH₂dihydroxycho- (SEQ ID NO: 8) lan-24-oic acid L283 — — — — DO3A- Gly(3β,5β 7a,12a)- f QWAVGHLNH- monoamide 3-amino-7,12- pentyldihydroxycho- (SEQ ID NO: 6) lan-24-oic acid L284 — — — — DO3A- Gly(3β,5β 7a,12a)- y QWAVBala- monoamide 3-amino-7,12- HF-Nle-dihydroxycho- NH₂ lan-24-oic acid (SEQ ID NO: 9) L285 — — — — DO3A- Gly(3β,5β 7a,12a)- f QWAVBala- monoamide 3-amino-7,12- HF-Nle-dihydroxycho- NH₂ lan-24-oic acid (SEQ ID NO: 9) L286 — — — — DO3A- Gly(3β,5β 7a,12a)- none QWAVGHFL- monoamide 3-amino-7,12- NH₂ dihydroxycho-(SEQ ID NO: 11) lan-24-oic acid L287 — — — — DO3A- Gly (3β,5β 7a,12a)-none QWAVGN- monoamide 3-amino-7,12- MeHis-L- dihydroxycho- M-NH₂lan-24-oic acid (SEQ ID NO: 16) L288 — — — — DO3A- Gly (3β,5β 7a,12a)-none LWAVGSFM- monoamide 3-amino-7,12- NH₂ dihydroxycho- (SEQ ID NO: 12)lan-24-oic acid L289 — — — — DO3A- Gly (3β,5β 7a,12a)- none HWAVGHLM-monoamide 3-amino-7,12- NH₂ dihydroxycho- (SEQ ID NO: 13) lan-24-oicacid L290 — — — — DO3A- Gly (3β,5β 7a,12a)- none LWATGHFM- monoamide3-amino-7,12- NH₂ dihydroxycho- (SEQ ID NO: 17) lan-24-oic acid L291 — —— — DO3A- Gly (3β,5β 7a,12a)- none QWAVGHFM- monoamide 3-amino-7,12- NH₂dihydroxycho- (SEQ ID NO: 14) lan-24-oic acid L292 — — — — DO3A- Gly3β,5β 7α,12α)- QRLGN QWAVGHLM- monoamide 3-amino-7,12- NH₂ dihydroxycho-(SEQ ID NO: 1) lan-24-oic acid L293 — — — — DO3A- Gly 3β,5β 7α,12α)-QRYGN QWAVGHLM- monoamide 3-amino-7,12- NH₂ dihydroxycho- (SEQ ID NO: 1)lan-24-oic acid L294 — — — — DO3A- Gly 3β,5β 7α,12α)- QKYGN QWAVGHLM-monoamide 3-amino-7,12- NH₂ dihydroxycho- (SEQ ID NO: 1) lan-24-oic acidL295 — — — — Pglu-Q-Lys Gly 3β,5β 7α,12α)- LG-N QWAVGHLM- (DO3A-3-amino-7,12- NH₂ monoamide) dihydroxycho- (SEQ ID NO: 1) lan-24-oicacid L304 — — — — DO3A- Gly 3-amino-3- none QRYGNQWAVGHLM- monoamidedeoxycholic NH₂ acid (SEQ ID NO: 4) L305 — — — — DO3A- Gly 3-amino-3-none QKYGNQWAVGHLM- monoamide deoxycholic NH₂ acid (SEQ ID NO: 5) L306DO3A- Gly 3-amino-3- none See FIG. 38 monoamide deoxycholic forstructure acid of targeting peptide

2D. Other Linking Groups

Other linking groups which may be used within the linker N—O—P include achemical group that serves to couple the GRP receptor targeting peptideto the metal chelator or optical label while not adversely affectingeither the targeting function of the GRP receptor targeting peptide orthe metal complexing function of the metal chelator or the detectabilityof the optical label. Suitable other linking groups include peptides(i.e., amino acids linked together) alone, a non-peptide group (e.g.,hydrocarbon chain) or a combination of an amino acid sequence and anon-peptide spacer.

In one embodiment, other linking groups for use within the linker N—O—Pinclude L-glutamine and hydrocarbon chains, or a combination thereof.

In another embodiment, other linking groups for use within the linkerN—O—P include a pure peptide linking group consisting of a series ofamino acids (e.g., diglycine, triglycine, gly-gly-glu, gly-ser-gly,etc.), in which the total number of atoms between the N-terminal residueof the GRP receptor targeting peptide and the metal chelator or theoptical label in the polymeric chain is ≦12 atoms.

In yet a further embodiment, other linking groups for use within thelinker N—O—P can also include a hydrocarbon chain [i.e.,R₁—(CH₂)_(n)—R₂] wherein n is 0-10, preferably n=3 to 9, R₁ is a group(e.g., H₂N—, HS—, —COOH) that can be used as a site for covalentlylinking the ligand backbone or the preformed metal chelator or metalcomplexing backbone or optical label; and R₂ is a group that is used forcovalent coupling to the N-terminal NH₂-group of the GRP receptortargeting peptide (e.g., R₂ is an activated COOH group). Severalchemical methods for conjugating ligands (i.e., chelators) or preferredmetal chelates to biomolecules have been well described in theliterature [Wilbur, 1992; Parker, 1990; Hermanson, 1996; Frizberg etal., 1995]. One or more of these methods could be used to link eitherthe uncomplexed ligand (chelator) or the radiometal chelate or opticallabel to the linker or to link the linker to the GRP receptor targetingpeptides. These methods include the formation of acid anhydrides,aldehydes, arylisothiocyanates, activated esters, orN-hydroxysuccinimides [Wilbur, 1992; Parker, 1990; Hermanson, 1996;Frizberg et al., 1995].

In a preferred embodiment, other linking groups for use within thelinker N—O—P may be formed from linker precursors having electrophilesor nucleophiles as set forth below:

-   -   LP1: a linker precursor having on at least two locations of the        linker the same electrophile E1 or the same nucleophile Nu1;    -   LP2: a linker precursor having an electrophile E1 and on another        location of the linker a different electrophile E2;    -   LP3: a linker precursor having a nucleophile Nu1 and on another        location of the linker a different nucleophile Nu2; or    -   LP4: a linker precursor having one end functionalized with an        electrophile E1 and the other with a nucleophile Nu1.

The preferred nucleophiles Nu1/Nu2 include —OH, —NH, —NR, —SH, —HN—NH₂,—RN—NH₂, and —RN—NHR′, in which R′ and R are independently selected fromthe definitions for R given above, but for R′ is not H.

The preferred electrophiles E1/E2 include —COOH, —CH═O (aldehyde),—CR═OR′ (ketone), —RN—C═S, —RN—C═O, —S—S-2-pyridyl, —SO₂—Y, —CH₂C(═O)Y,and

wherein Y can be selected from the following groups:

3. GRP Receptor Targeting Peptide

The GRP receptor targeting peptide (i.e., G in the formula M—N—O—P—G) isany peptide, equivalent, derivative or analogue thereof which has abinding affinity for the GRP receptor family.

The GRP receptor targeting peptide may take the form of an agonist or anantagonist. A GRP receptor targeting peptide agonist is known to“activate” the cell following binding with high affinity and may beinternalized by the cell. Conversely, GRP receptor targeting peptideantagonists are known to bind only to the GRP receptor on the cellwithout being internalized by the cell and without “activating” thecell. In a preferred embodiment, the GRP receptor targeting peptide isan agonist.

In a more preferred embodiment of the present invention, the GRP agonistis a bombesin (BBN) analogue and/or a derivative thereof. The BBNderivative or analog thereof preferably contains either the same primarystructure of the BBN binding region (i.e., BBN(7-14) [SEQ ID NO:1]) orsimilar primary structures, with specific amino acid substitutions thatwill specifically bind to GRP receptors with better or similar bindingaffinities as BBN alone (i.e., Kd<25 nM). Suitable compounds includepeptides, peptidomimetics and analogues and derivatives thereof. Thepresence of L-methionine (Met) at position BBN-14 will generally conferagonistic properties while the absence of this residue at BBN-14generally confers antagonistic properties [Hoffken, 1994]. Some usefulbombesin analogues are disclosed in U.S. Patent Pub. 2003/0224998,incorporated here in its entirety.

It is well documented in the art that there are a few and selectivenumber of specific amino acid substitutions in the BBN (8-14) bindingregion (e.g., D-Ala¹¹ for L-Gly¹¹ or D-Trp⁸ for L-Trp⁸), which can bemade without decreasing binding affinity [Leban et al., 1994; Qin etal., 1994; Jensen et al., 1993]. In addition, attachment of some aminoacid chains or other groups to the N-terminal amine group at positionBBN-8 (i.e., the Trp⁸ residue) can dramatically decrease the bindingaffinity of BBN analogues to GRP receptors [Davis et al., 1992; Hoffken,1994; Moody et al., 1996; Coy, et al., 1988; Cai et al., 1994]. In a fewcases, it is possible to append additional amino acids or chemicalmoieties without decreasing binding affinity.

Analogues of BBN receptor targeting peptides include molecules thattarget the GRP receptors with avidity that is greater than or equal toBBN, as well as muteins, retropeptides and retro-inverso-peptides of GRPor BBN. One of ordinary skill will appreciate that these analogues mayalso contain modifications which include substitutions, and/or deletionsand/or additions of one or several amino acids, insofar that thesemodifications do not negatively alter the biological activity of thepeptides described therein. These substitutions may be carried out byreplacing one or more amino acids by their synonymous amino acids.Synonymous amino acids within a group are defined as amino acids thathave sufficient physicochemical properties to allow substitution betweenmembers of a group in order to preserve the biological function of themolecule.

Deletions or insertions of amino acids may also be introduced into thedefined sequences provided they do not alter the biological functions ofsaid sequences. Preferentially such insertions or deletions should belimited to 1, 2, 3, 4 or 5 amino acids and should not remove orphysically disturb or displace amino acids which are critical to thefunctional conformation. Muteins of the GRP receptor targeting peptidesdescribed herein may have a sequence homologous to the sequencedisclosed in the present specification in which amino acidsubstitutions, deletions, or insertions are present at one or more aminoacid positions. Muteins may have a biological activity that is at least40%, preferably at least 50%, more preferably 60-70%, most preferably80-90% of the peptides described herein. However, they may also have abiological activity greater than the peptides specifically exemplified,and thus do not necessarily have to be identical to the biologicalfunction of the exemplified peptides. Analogues of GRP receptortargeting peptides also include peptidomimetics or pseudopeptidesincorporating changes to the amide bonds of the peptide backbone,including thioamides, methylene amines, and E-olefins. Also peptidesbased on the structure of GRP, BBN or their peptide analogues with aminoacids replaced by N-substituted hydrazine carbonyl compounds (also knownas aza amino acids) are included in the term analogues as used herein.

The GRP receptor targeting peptide can be prepared by various methodsdepending upon the selected chelator. The peptide can generally be mostconveniently prepared by techniques generally established and known inthe art of peptide synthesis, such as the solid-phase peptide synthesis(SPPS) approach. Solid-phase peptide synthesis (SPPS) involves thestepwise addition of amino acid residues to a growing peptide chain thatis linked to an insoluble support or matrix, such as polystyrene. TheC-terminal residue of the peptide is first anchored to a commerciallyavailable support with its amino group protected with an N-protectingagent such as a t-butyloxycarbonyl group (Boc) or afluorenylmethoxycarbonyl (Fmoc) group. The amino protecting group isremoved with suitable deprotecting agents such as TFA in the case of Bocor piperidine for Fmoc and the next amino acid residue (in N-protectedform) is added with a coupling agent such asN,N′-dicyclohexylcarbodiimide (DCC), or N,N′-diisopropylcarbodiimide(DIC) or 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU). Upon formation of a peptide bond, thereagents are washed from the support. After addition of the finalresidue, the peptide is cleaved from the support with a suitable reagentsuch as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).

The linker may then be coupled to form a conjugate by reacting the freeamino group of the Trp⁸ residue of the GRP receptor targeting peptidewith an appropriate functional group of the linker. The entire constructof chelator, linker and targeting moiety discussed above may also beassembled on resin and then cleaved by agency of suitable reagents suchas trifluoroacetic acid or HF, as well.

Bombesin (7-14) is subject to proteolytic cleavage in vitro and in vivo,which shortens the half-life of the peptide. It is well known in theliterature that the amide bond of the backbone of the polypeptide may besubstituted and retain activity, while resisting proteolytic cleavage.For example, to reduce or eliminate undesired proteolysis, or otherdegradation pathways that diminish serum stability, resulting in reducedor abolished bioactivity, or to restrict or increase conformationalflexibility, it is common to substitute amide bonds within the backboneof the peptides with functionality that mimics the existing conformationor alters the conformation in the manner desired. Such modifications mayproduce increased binding affinity or improved pharmacokinetic behavior.It is understood that those knowledgeable in the art of peptidesynthesis can make the following amide bond-changes for any amide bondconnecting two amino acids (e.g., amide bonds in the targeting moiety,linker, chelator, etc.) with the expectation that the resulting peptidescould have the same or improved activity: insertion ofalpha-N-methylamides or backbone thioamides, removal of the carbonyl toproduce the cognate secondary amines, replacement of one amino acid withan aza-aminoacid to produce semicarbazone derivatives, and use ofE-olefins and substituted E-olefins as amide bond surrogates. Thehydrolysis can also be prevented by incorporation of a D-amino acid ofone of the amino acids of the labile amide bond, or by alpha-methylaminoacid derivatives. Backbone amide bonds have also been replaced byheterocycles such as oxazoles, pyrrolidinones, imidazoles, as well asketomethylenes and fluoroolefins.

Some specific compounds including such amide bond modifications arelisted in Table 4a. The abbreviations used in Table 4a for the variousamide bond modifications are exemplified below:

TABLE 4A

Preferred Amide Bond Modified Analogs Com- pound M—N—O—P BBN AnalogueL401 DO3A-mono- Nme-Q W A V G H L M-NH₂ amide-G-Abz4 L402 DO3A-mono-Q-Ψ[CSNH] W A V G H L M-NH₂ amide-G-Abz4 L403 DO3A-mono- Q-Ψ[CH₂NH] W AV G H L M-NH₂ amide-G-Abz4 L404 DO3A-mono- Q-Ψ[CH═CH] W A V G H L M-NH₂amide-G-Abz4 L405 DO3A-mono- α-MeQ W A V G H L M-NH₂ amide-G-Abz4 L406DO3A-mono- Q Nme-W A V G H L M-NH₂ amide-G-Abz4 L407 DO3A-mono- QW-Ψ[CSNH] A V G H L M-NH₂ amide-G-Abz4 L408 DO3A-mono- Q W-Ψ[CH₂NH] A VG H L M-NH₂ amide-G-Abz4 L409 DO3A-mono- Q W-Ψ[CH═CH] A V G H L M-NH₂amide-G-Abz4 L410 DO3A-mono- Q α-MeW A V G H L M-NH₂ amide-G-Abz4 L411DO3A-mono- Q W Nme-A V G H L M-NH₂ amide-G-Abz4 L412 DO3A-mono- Q WA-Ψ[CSNH] V G H L M-NH₂ amide-G-Abz4 L413 DO3A-mono- Q W A-Ψ[CH₂NH] V GH L M-NH₂ amide-G-Abz4 L414 DO3A-mono- Q W Aib V G H L M-NH₂amide-G-Abz4 L415 DO3A-mono- Q W A V Sar H L M-NH₂ amide-G-Abz4 L416DO3A-mono- Q W A V G-Ψ[CSNH] H L M-NH₂ amide-G-Abz4 L417 DO3A-mono- Q WA V G-Ψ[CH═CH] H L M-NH₂ amide-G-Abz4 L418 DO3A-mono- Q W A V Dala H LM-NH₂ amide-G-Abz4 L419 DO3A-mono- Q W A V G Nme-His L M-NH₂amide-G-Abz4 L420 DO3A-mono- Q W A V G H-Ψ[CSNH] L M-NH₂ amide-G-Abz4L421 DO3A-mono- Q W A V G H-Ψ[CH₂NH] L M-NH₂ amide-G-Abz4 L422DO3A-mono- Q W A V G H-Ψ[CH═CH] L M-NH₂ amide-G-Abz4 L423 DO3A-mono- Q WA V G α-MeH L M-NH₂ amide-G-Abz4 L424 DO3A-mono- Q W A V G H Nme-L M-NH₂amide-G-Abz4 L425 DO3A-mono- Q W A V G H α-MeL M-NH₂ amide-G-Abz4 L300DO3A-mono- Q W A V G H F–L NH₂ amide-G-ABz4

4. Labeling and Administration of Radiopharmaceutical Compounds

Incorporation of the metal within the radiopharmaceutical conjugates canbe achieved by various methods commonly known in the art of coordinationchemistry. When the metal is ^(99m)Tc, a preferred radionuclide fordiagnostic imaging, the following general procedure can be used to forma technetium complex. A peptide-chelator conjugate solution is formed byinitially dissolving the conjugate in water, dilute acid, or in anaqueous solution of an alcohol such as ethanol. The solution is thenoptionally degassed to remove dissolved oxygen. When an —SH group ispresent in the peptide, a thiol protecting group such as Acm(acetamidomethyl), trityl or other thiol protecting group may optionallybe used to protect the thiol from oxidation. The thiol protectinggroup(s) are removed with a suitable reagent, for example with sodiumhydroxide, and are then neutralized with an organic acid such as aceticacid (pH 6.0-6.5). Alternatively, the thiol protecting group can beremoved in situ during technetium chelation. In the labeling step,sodium pertechnetate obtained from a molybdenum generator is added to asolution of the conjugate with a sufficient amount of a reducing agent,such as stannous chloride, to reduce technetium and is either allowed tostand at room temperature or is heated. The labeled conjugate can beseparated from the contaminants ^(99m)TcO₄ ⁻ and colloidal ^(99m)TcO₂chromatographically, for example with a C-18 Sep Pak cartridge[Millipore Corporation, Waters Chromatography Division, 34 Maple Street,Milford, Mass. 01757] or by HPLC using methods known to those skilled inthe art.

In an alternative method, the labeling can be accomplished by atranschelation reaction. In this method, the technetium source is asolution of technetium that is reduced and complexed with labile ligandsprior to reaction with the selected chelator, thus facilitating ligandexchange with the selected chelator. Examples of suitable ligands fortranschelation includes tartrate, citrate, gluconate, andheptagluconate. It will be appreciated that the conjugate can be labeledusing the techniques described above, or alternatively, the chelatoritself may be labeled and subsequently coupled to the peptide to formthe conjugate; a process referred to as the “prelabeled chelate” method.Re and Tc are both in row VIIB of the Periodic Table and they arechemical congeners. Thus, for the most part, the complexation chemistryof these two metals with ligand frameworks that exhibit high in vitroand in vivo stabilities are the same [Eckelman, 1995] and similarchelators and procedures can be used to label with Re. Many ^(99m)Tc or^(186/188)Re complexes, which are employed to form stable radiometalcomplexes with peptides and proteins, chelate these metals in their +5oxidation state [Lister-James et al., 1997]. This oxidation state makesit possible to selectively place ^(99m)Tc- or ^(186/188)Re into ligandframeworks already conjugated to the biomolecule, constructed from avariety of ^(99m)Tc(V) and/or ^(186/188)Re(V) weak chelates (e.g.,^(99m)Tc-glucoheptonate, citrate, gluconate, etc.) [Eckelman, 1995;Lister-James et al., 1997; Pollak et al., 1996]. These references arehereby incorporated by reference in their entirety.

5. Diagnostic and Therapeutic Uses

When labeled with diagnostically and/or therapeutically useful metals oroptical labels, compounds of the present invention can be used to treatand/or detect any pathology involving overexpression of GRP receptors(or NMB receptors) by procedures established in the art ofradiodiagnostics, radiotherapeutics and optical imaging. [See, e.g.,Bushbaum, 1995; Fischman et al., 1993; Schubiger et al., 1996; Lowbertzet al., 1994; Krenning et al., 1994; examples of optical dyes include,but are not limited to those described in WO 98/18497, WO 98/18496, WO98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO96/23524, WO 98/47538, and references cited therein, hereby incorporatedby reference in their entirety.]

GRP-R expression is highly upregulated in a variety of human tumors. Seee.g., WO 99/62563. Thus, compounds of the invention may be widely usefulin treating and diagnosing cancers, including prostate cancer (primaryand metastatic), breast cancer (primary and metastatic), colon cancer,gastric cancer, pancreatic cancer, non small cell lung cancer, smallcell lung cancer, gastrinomas, melanomas, glioblastomas, neuroblastomas,uterus leiomyosarcoma tumors, prostatic intraepithelial neoplasias[PIN], and ovarian cancer. Additionally, compounds of the invention maybe useful to distinguish between conditions in which GRP receptors areupregulated and those in which they are not (e.g. chronic pancreatitisand ductal pancreatic carcinoma, respectively

The compounds of the invention, which, as explained in more detail inthe Examples, show greater specificity and higher uptake in tumors invivo than compounds without the novel linkers disclosed herein, exhibitan improved ability to target GRP receptor-expressing tumors and thus toimage or deliver radiotherapy to these tissues. Indeed, as shown in theExamples, radiotherapy is more effective (and survival time increased)using compounds of the invention.

The diagnostic application of these compounds can be as a first linediagnostic screen for the presence of neoplastic cells usingscintigraphic, optical, sonoluminescence or photoacoustic imaging, as anagent for targeting neoplastic tissue using hand-held radiationdetection instrumentation in the field of radioimmuno guided surgery(RIGS), as a means to obtain dosimetry data prior to administration ofthe matched pair radiotherapeutic compound, and as a means to assess GRPreceptor population as a function of treatment over time.

The therapeutic application of these compounds can be defined as anagent that will be used as a first line therapy in the treatment ofcancer, as combination therapy where these agents could be utilized inconjunction with adjuvant chemotherapy, and/or as a matched pairtherapeutic agent. The matched pair concept refers to a singleunmetallated compound which can serve as both a diagnostic and atherapeutic agent depending on the radiometal that has been selected forbinding to the appropriate chelate. If the chelator cannot accommodatethe desired metals, appropriate substitutions can be made to accommodatethe different metal while maintaining the pharmacology such that thebehavior of the diagnostic compound in vivo can be used to predict thebehavior of the radiotherapeutic compound. When utilized in conjunctionwith adjuvant chemotherapy any suitable chemotherapeutic may be used,including for example, antineoplastic agents, such as platinum compounds(e.g., spiroplatin, cisplatin, and carboplatin), methotrexate,adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside,arabinosyl adenine, mercaptopolylysine, vincristine, busulfan,chlorambucil, melphalan (e.g., PAM, a, L-PAM or phennylalanine mustard),mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin(actinomycin D), daunorubcin hydrochloride, doxorubicin hydrochloride,taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrolacetate, tamoxifen citrate, testolactone, trilostane, amsacrine(m-AMSA), asparaginase (L-asparaginase) Erwina aparaginase,etoposide-(VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26),vinblastine sulfate (VLB), and arabinosyl. In certain embodiments, thetherapeutic may be monoclonal antibody, such as a monoclonal antibodycapable of binding to melanoma antigen.

A conjugate labeled with a radionuclide metal, such as ^(99m)Tc, can beadministered to a mammal, including human patients or subjects, by, forexample, intravenous, subcutaneous or intraperitoneal injection in apharmaceutically acceptable carrier and/or solution such as saltsolutions like isotonic saline. Radiolabeled scintigraphic imagingagents provided by the present invention are provided having a suitableamount of radioactivity. In forming ^(99m)Tc radioactive complexes, itis generally preferred to form radioactive complexes in solutionscontaining radioactivity at concentrations of from about 0.01 millicurie(mCi) to 100 mCi per mL. Generally, the unit dose to be administered hasa radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to30 mCi. The solution to be injected at unit dosage is from about 0.01 mLto about 10 mL. The amount of labeled conjugate appropriate foradministration is dependent upon the distribution profile of the chosenconjugate in the sense that a rapidly cleared conjugate may need to beadministered in higher doses than one that clears less rapidly. In vivodistribution and localization can be tracked by standard scintigraphictechniques at an appropriate time subsequent to administration;typically between thirty minutes and 180 minutes depending upon the rateof accumulation at the target site with respect to the rate of clearanceat non-target tissue. For example, after injection of the diagnosticradionuclide-labeled compounds of the invention into the patient, agamma camera calibrated for the gamma ray energy of the nuclideincorporated in the imaging agent can be used to image areas of uptakeof the agent and quantify the amount of radioactivity present in thesite. Imaging of the site in vivo can take place in a few minutes.However, imaging can take place, if desired, hours or even longer, afterthe radiolabeled peptide is injected into a patient. In most instances,a sufficient amount of the administered dose will accumulate in the areato be imaged within about 0.1 hour to permit the taking of scintiphotos.

The compounds of the present invention can be administered to a patientalone or as part of a composition that contains other components such asexcipients, diluents, radical scavengers, stabilizers, and carriers, allof which are well-known in the art. The compounds can be administered topatients either intravenously or intraperitoneally.

There are numerous advantages associated with the present invention. Thecompounds made in accordance with the present invention form stable,well-defined ^(99m)Tc or ^(186/188)Re labeled compounds. Similarcompounds of the invention can also be made by using appropriatechelator frameworks for the respective radiometals, to form stable,well-defined products labeled with ¹⁵³Sm, ⁹⁰Y, ¹⁶⁶Ho, ¹⁰⁵Rh, ¹⁹⁹Au,¹⁴⁹Pm, ¹⁷⁷Lu, ¹¹¹In or other radiometals. The radiolabeled GRP receptortargeting peptides selectively bind to neoplastic cells expressing GRPreceptors, and if an agonist is used, become internalized, and areretained in the tumor cells for extended time periods. The radioactivematerial that does not reach (i.e., does not bind) the cancer cells ispreferentially excreted efficiently into the urine with minimalretention of the radiometal in the kidneys.

6. Optical Imaging, Sonoluminescence, Photoacoustic Imaging andPhototherapy

In accordance with the present invention, a number of optical parametersmay be employed to determine the location of a target with in vivo lightimaging after injection of the subject with an optically-labeledcompound of the invention. Optical parameters to be detected in thepreparation of an image may include transmitted radiation, absorption,fluorescent or phosphorescent emission, light reflection, changes inabsorbance amplitude or maxima, and elastically scattered radiation. Forexample, biological tissue is relatively translucent to light in thenear infrared (NIR) wavelength range of 650-1000 nm. NIR radiation canpenetrate tissue up to several centimeters, permitting the use ofcompounds of the present invention to image target-containing tissue invivo. The use of visible and near-infrared (NIR) light in clinicalpractice is growing rapidly. Compounds absorbing or emitting in thevisible, NIR, or long-wavelength (UV-A, >350 nm) region of theelectromagnetic spectrum are potentially useful for optical tomographicimaging, endoscopic visualization, and phototherapy.

A major advantage of biomedical optics lies in its therapeuticpotential. Phototherapy has been demonstrated to be a safe and effectiveprocedure for the treatment of various surface lesions, both externaland internal. Dyes are important to enhance signal detection and/orphotosensitizing of tissues in optical imaging and phototherapy.Previous studies have shown that certain dyes can localize in tumors andserve as a powerful probe for the detection and treatment of smallcancers (D. A. Bellnier et al., Murine pharmacokinetics and antitumorefficacy of the photodynamic sensitizer 2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a, J. Photochem. Photobiol., 1993, 20, pp. 55-61; G. A.Wagnieres et al., In vivo fluorescence spectroscopy and imaging foroncological applications, Photochem. Photobiol., 1998, 68, pp. 603-632;J. S. Reynolds et al., Imaging of spontaneous canine mammary tumorsusing fluorescent contrast agents, Photochem. Photobiol., 1999, 70, pp.87-94). All of these listed references are hereby incorporated byreference in their entirety. However, these dyes do not localizepreferentially in malignant tissues.

In an exemplary embodiment, the compounds of the invention may beconjugated with photolabels, such as optical dyes, including organicchromophores or fluorophores, having extensive delocalized ring systemsand having absorption or emission maxima in the range of 400-1500 nm.The compounds of the invention may alternatively be derivatized with abioluminescent molecule. The preferred range of absorption maxima forphotolabels is between 600 and 1000 nm to minimize interference with thesignal from hemoglobin. Preferably, photoabsorption labels have largemolar absorptivities, e.g. >10⁵ cm⁻¹ M⁻¹, while fluorescent optical dyeswill have high quantum yields. Examples of optical dyes include, but arenot limited to those described in U.S. Pat. No. 6,641,798, WO 98/18497,WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO97/18841, WO 96/23524, WO 98/47538, and references cited therein, allhereby incorporated by reference in their entirety. For example, thephotolabels may be covalently linked directly to compounds of theinvention, such as, for example, compounds comprised of GRP receptortargeting peptides and linkers of the invention. Several dyes thatabsorb and emit light in the visible and near-infrared region ofelectromagnetic spectrum are currently being used for various biomedicalapplications due to their biocompatibility, high molar absorptivity,and/or high fluorescence quantum yields. The high sensitivity of theoptical modality in conjunction with dyes as contrast agents parallelsthat of nuclear medicine, and permits visualization of organs andtissues without the undesirable effect of ionizing radiation. Cyaninedyes with intense absorption and emission in the near-infrared (NIR)region are particularly useful because biological tissues are opticallytransparent in this region (B. C. Wilson, Optical properties of tissues.Encyclopedia of Human Biology, 1991, 5, 587-597). For example,indocyanine green, which absorbs and emits in the NIR region has beenused for monitoring cardiac output, hepatic functions, and liver bloodflow (Y-L. He, H. Tanigami, H. Ueyama, T. Mashimo, and I. Yoshiya,Measurement of blood volume using indocyanine green measured withpulse-spectrometry: Its reproducibility and reliability. Critical CareMedicine, 1998, 26(8), 1446-1451; J. Caesar, S. Shaldon, L. Chiandussi,et al., The use of Indocyanine green in the measurement of hepatic bloodflow and as a test of hepatic function. Clin. Sci. 1961, 21, 43-57) andits functionalized derivatives have been used to conjugate biomoleculesfor diagnostic purposes (R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, etal., Cyanine dye labeling reagents: Sulfoindocyanine succinimidylesters. Bioconjugate Chemistry, 1993, 4(2), 105-111; Linda G. Lee andSam L. Woo. “N-Heteroaromatic ion and iminium ion substituted cyaninedyes for use as fluorescent labels”, U.S. Pat. No. 5,453,505; EricHohenschuh, et al. “Light imaging contrast agents”, WO 98/48846;Jonathan Turner, et al. “Optical diagnostic agents for the diagnosis ofneurodegenerative diseases by means of near infra-red radiation”, WO98/22146; Kai Licha, et al. “In-vivo diagnostic process by near infraredradiation”, WO 96/17628; Robert A. Snow, et al., Compounds, WO 98/48838,U.S. Pat. No. 6,641,798. All of these listed references are herebyincorporated by reference in their entirety.

After injection of the optically-labeled compound, the patient isscanned with one or more light sources (e.g., a laser) in the wavelengthrange appropriate for the photolabel employed in the agent. The lightused may be monochromatic or polychromatic and continuous or pulsed.Transmitted, scattered, or reflected light is detected via aphotodetector tuned to one or multiple wavelengths to determine thelocation of target-containing tissue (e.g., tissue containing GRP) inthe subject. Changes in the optical parameter may be monitored over timeto detect accumulation of the optically-labeled reagent at the targetsite (e.g. the tumor or other site with GRP receptors). Standard imageprocessing and detecting devices may be used in conjunction with theoptical imaging reagents of the present invention.

The optical imaging reagents described above may also be used foracousto-optical or sonoluminescent imaging performed withoptically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO98/57666, and references therein). In acousto-optical imaging,ultrasound radiation is applied to the subject and affects the opticalparameters of the transmitted, emitted, or reflected light. Insonoluminescent imaging, the applied ultrasound actually generates thelight detected. Suitable imaging methods using such techniques aredescribed in WO 98/57666.

Various imaging techniques and reagents are described in U.S. Pat. Nos.6,663,847, 6,656,451, 6,641,798, 6,485,704, 6,423,547, 6,395,257,6,280,703, 6,277,841, 6,264,920, 6,264,919, 6,228,344, 6,217,848,6,190,641, 6,183,726, 6,180,087, 6,180,086, 6,180,085, 6,013,243, andpublished U.S. Patent Applications 2003185756, 20031656432, 2003158127,2003152577, 2003143159, 2003105300, 2003105299, 2003072763, 2003036538,2003031627, 2003017164, 2002169107, 2002164287, and 2002156117, all ofwhich are hereby incorporated by reference.

7. Radiotherapy

Radioisotope therapy involves the administration of a radiolabeledcompound in sufficient quantity to damage or destroy the targetedtissue. After administration of the compound (by e.g., intravenous,subcutaneous, or intraperitonal injection), the radiolabeledpharmaceutical localizes preferentially at the disease site (in thisinstance, tumor tissue or other tissue that expresses the pertinent GRPreceptor). Once localized, the radiolabeled compound then damages ordestroys the diseased tissue with the energy that is released during theradioactive decay of the isotope that is administered. As discussedherein, the invention also encompasses use of radiotherapy incombination with adjuvant chemotherapy (or in combination with any otherappropriate therapeutic agent).

The design of a successful radiotherapeutic involves several criticalfactors:

-   -   1. selection of an appropriate targeting group to deliver the        radioactivity to the disease site;    -   2. selection of an appropriate radionuclide that releases        sufficient energy to damage that disease site, without        substantially damaging adjacent normal tissues; and    -   3. selection of an appropriate combination of the targeting        group and the radionuclide without adversely affecting the        ability of this conjugate to localize at the disease site. For        radiometals, this often involves a chelating group that        coordinates tightly to the radionuclide, combined with a linker        that couples said chelate to the targeting group, and that        affects the overall biodistribution of the compound to maximize        uptake in target tissues and minimize uptake in normal,        non-target organs.

The present invention provides radiotherapeutic agents that satisfy allthree of the above criteria, through proper selection of targetinggroup, radionuclide, metal chelate and linker.

Radiotherapeutic agents may contain a chelated 3+ metal ion from theclass of elements known as the lanthanides (elements of atomic number57-71) and their analogs (i.e. M³⁺ metals such as yttrium and indium).Typical radioactive metals in this class include the isotopes90-Yttrium, 111-Indium, 149-Promethium, 153-Samarium, 166-Dysprosium,166-Holmium, 175-Ytterbium, and ¹⁷⁷-Lutetium. All of these metals (andothers in the lanthanide series) have very similar chemistries, in thatthey remain in the +3 oxidation state, and prefer to chelate to ligandsthat bear hard (oxygen/nitrogen) donor atoms, as typified by derivativesof the well known chelate DTPA (diethylenetriaminepentaacetic acid) andpolyaza-polycarboxylate macrocycles such as DOTA(1,4,7,10-tetrazacyclododecane-N,N′,N″,N′″-tetraacetic acid and itsclose analogs. The structures of these chelating ligands, in their fullydeprotonated form are shown below.

These chelating ligands encapsulate the radiometal by binding to it viamultiple nitrogen and oxygen atoms, thus preventing the release of free(unbound) radiometal into the body. This is important, as in vivodissociation of 3⁺ radiometals from their chelate can result in uptakeof the radiometal in the liver, bone and spleen [Brechbiel M W, Gansow OA, “Backbone-substituted DTPA ligands for ⁹⁰Y radioimmunotherapy”,Bioconj. Chem. 1991; 2: 187-194; Li, W P, Ma D S, Higginbotham C,Hoffman T, Ketring A R, Cutler C S, Jurisson, S S, “Development of an invitro model for assessing the in vivo stability of lanthanide chelates.”Nucl. Med. Biol. 2001; 28(2): 145-154; Kasokat T, Urich K.Arzneim.-Forsch, “Quantification of dechelation of gadopentetatedimeglumine in rats”. 1992; 42(6): 869-763. Unless one is specificallytargeting these organs, such non-specific uptake is highly undesirable,as it leads to non-specific irradiation of non-target tissues, which canlead to such problems as hematopoietic suppression due to irradiation ofbone marrow.

For radiotherapy applications any of the chelators for therapeuticradionuclides disclosed herein may be used. However, forms of the DOTAchelate [Tweedle M F, Gaughan G T, Hagan J T,“1-Substituted-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecaneand analogs.” U.S. Pat. No. 4,885,363, Dec. 5, 1989] are particularlypreferred, as the DOTA chelate is expected to de-chelate less in thebody than DTPA or other linear chelates. Compounds L64 and L70 (whenlabeled with an appropriate therapeutic radionuclide) are particularlypreferred for radiotherapy.

General methods for coupling DOTA-type macrocycles to targeting groupsthrough a linker (e.g. by activation of one of the carboxylates of theDOTA to form an active ester, which is then reacted with an amino groupon the linker to form a stable amide bond), are known to those skilledin the art. (See e.g. Tweedle et al. U.S. Pat. No. 4,885,363). Couplingcan also be performed on DOTA-type macrocycles that are modified on thebackbone of the polyaza ring.

The selection of a proper nuclide for use in a particularradiotherapeutic application depends on many factors, including:

a. Physical half-life—This should be long enough to allow synthesis andpurification of the radiotherapeutic construct from radiometal andconjugate, and delivery of said construct to the site of injection,without significant radioactive decay prior to injection. Preferably,the radionuclide should have a physical half-life between about 0.5 and8 days.

b. Energy of the emission(s) from the radionuclide—Radionuclides thatare particle emitters (such as alpha emitters, beta emitters and Augerelectron emitters) are particularly useful, as they emit highlyenergetic particles that deposit their energy over short distances,thereby producing highly localized damage. Beta emitting radionuclidesare particularly preferred, as the energy from beta particle emissionsfrom these isotopes is deposited within 5 to about 150 cell diameters.Radiotherapeutic agents prepared from these nuclides are capable ofkilling diseased cells that are relatively close to their site oflocalization, but cannot travel long distances to damage adjacent normaltissue such as bone marrow.

c. Specific activity (i.e. radioactivity per mass of theradionuclide)-Radionuclides that have high specific activity (e.g.generator produced 90-Y, 111-In, 177-Lu) are particularly preferred. Thespecific activity of a radionuclide is determined by its method ofproduction, the particular target that is used to produce it, and theproperties of the isotope in question.

Many of the lanthanides and lanthanoids include radioisotopes that havenuclear properties that make them suitable for use as radiotherapeuticagents, as they emit beta particles. Some of these are listed in thetable below.

Approximate range of b- Half-Life Max b- energy Gamma energy particleIsotope (days) (MeV) (keV) (cell diameters) ¹⁴⁹-Pm 2.21 1.1 286 60¹⁵³-Sm 1.93 0.69 103 30 ¹⁶⁶-Dy 3.40 0.40 82.5 15 ¹⁶⁶-Ho 1.12 1.8 80.6117 ¹⁷⁵-Yb 4.19 0.47 396 17 ¹⁷⁷-Lu 6.71 0.50 208 20 ⁹⁰-Y 2.67 2.28 — 150¹¹¹-In 2.810 Auger electron 173,247  <5 μm emitter Pm: Promethium, Sm:Samarium, Dy: Dysprosium, Ho: Holmium, Yb: Ytterbium, Lu: Lutetium, Y:Yttrium, In: Indium

Methods for the preparation of radiometals such as beta-emittinglanthanide radioisotopes are known to those skilled in the art, and havebeen described elsewhere [e.g., Cutler C S, Smith C J, Ehrhardt G J.;Tyler T T, Jurisson S S, Deutsch E. “Current and potential therapeuticuses of lanthanide radioisotopes.” Cancer Biother. Radiopharm. 2000;15(6): 531-545]. Many of these isotopes can be produced in high yieldfor relatively low cost, and many (e.g. ⁹⁰—Y, ¹⁴⁹—Pm, ¹⁷⁷—Lu) can beproduced at close to carrier-free specific activities (i.e. the vastmajority of atoms are radioactive). Since non-radioactive atoms cancompete with their radioactive analogs for binding to receptors on thetarget tissue, the use of high specific activity radioisotope isimportant, to allow delivery of as high a dose of radioactivity to thetarget tissue as possible.

Radiotherapeutic derivatives of the invention containing beta-emittingisotopes of rhenium (¹⁸⁶-Re and ¹⁸⁸-Re) are also particularly preferred.

8. Dosages And Additives

Proper dose schedules for the compounds of the present invention areknown to those skilled in the art. The compounds can be administeredusing many methods which include, but are not limited to, a single ormultiple IV or IP injections. For radiopharmaceuticals, one administersa quantity of radioactivity that is sufficient to permit imaging or, inthe case of radiotherapy, to cause damage or ablation of the targetedGRP-R bearing tissue, but not so much that substantive damage is causedto non-target (normal tissue). The quantity and dose required forscintigraphic imaging is discussed supra. The quantity and dose requiredfor radiotherapy is also different for different constructs, dependingon the energy and half-life of the isotope used, the degree of uptakeand clearance of the agent from the body and the mass of the tumor. Ingeneral, doses can range from a single dose of about 30-50 mCi to acumulative dose of up to about 3 Curies.

For optical imaging compounds, dosages sufficient to achieve the desiredimage enhancement are known to those skilled in the art and may varywidely depending on the dye or other compound used, the organ or tissueto be imaged, the imaging equipment used, etc.

The compositions of the invention can include physiologically acceptablebuffers, and can require radiation stabilizers to prevent radiolyticdamage to the compound prior to injection. Radiation stabilizers areknown to those skilled in the art, and may include, for example,para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.

A single, or multi-vial kit that contains all of the components neededto prepare the diagnostic or therapeutic agents of this invention is anintegral part of this invention. In the case of radiopharmaceuticals,such kits will often include all necessary ingredients except theradionuclide.

For example, a single-vial kit for preparing a radiopharmaceutical ofthe invention preferably contains a chelator/linker/targeting peptideconjugate of the formula M—N—O—P—G, a source of stannous salt (ifreduction is required, e.g., when using technetium), or otherpharmaceutically acceptable reducing agent, and is appropriatelybuffered with pharmaceutically acceptable acid or base to adjust the pHto a value of about 3 to about 9. The quantity and type of reducingagent used will depend highly on the nature of the exchange complex tobe formed. The proper conditions are well known to those that areskilled in the art. It is preferred that the kit contents be inlyophilized form. Such a single vial kit may optionally contain labileor exchange ligands such as glucoheptonate, gluconate, mannitol, malate,citric or tartaric acid and can also contain reaction modifiers such asdiethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraaceticacid (EDTA), or α, β, or γ-cyclodextrin that serve to improve theradiochemical purity and stability of the final product. The kit mayalso contain stabilizers, bulking agents such as mannitol, that aredesigned to aid in the freeze-drying process, and other additives knownto those skilled in the art.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial may contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g. the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains thechelator and targeting peptide, as well as buffers appropriate to adjustthe pH to its optimal value. After a reaction time of about 5 to 60minutes, the complexes of the present invention are formed. It isadvantageous that the contents of both vials of this multi-vial kit belyophilized. As above, reaction modifiers, exchange ligands,stabilizers, bulking agents, etc. may be present in either or bothvials.

General Preparation of Compounds

The compounds of the present invention can be prepared by variousmethods depending upon the selected chelator. The peptide portion of thecompound can be most conveniently prepared by techniques generallyestablished and known in the art of peptide synthesis, such as thesolid-phase peptide synthesis (SPPS) approach. Because it is amenable tosolid phase synthesis, employing alternating FMOC protection anddeprotection is the preferred method of making short peptides.Recombinant DNA technology is preferred for producing proteins and longfragments thereof.

Solid-phase peptide synthesis (SPPS) involves the stepwise addition ofamino acid residues to a growing peptide chain that is linked to aninsoluble support or matrix, such as polystyrene. The C-terminal residueof the peptide is first anchored to a commercially available supportwith its amino group protected with an N-protecting agent such as at-butyloxycarbonyl group (Boc) or a fluorenylmethoxycarbonyl (Fmoc)group. The amino protecting group is removed with suitable deprotectingagents such as TFA in the case of Boc or piperidine for Fmoc and thenext amino acid residue (in N-protected form) is added with a couplingagent such as diisopropylcarbodiimide (DIC). Upon formation of a peptidebond, the reagents are washed from the support. After addition of thefinal residue, the peptide is cleaved from the support with a suitablereagent such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).

Alternative Preparation of the Compounds Via Segment Coupling

The compounds of the invention may also be prepared by the process knownin the art as segment coupling or fragment condensation (Barlos, K. andGatos, D.; 2002 “Convergent Peptide Synthesis” in Fmoc Solid PhaseSynthesis—A Practical Approach; Eds. Chan, W. C. and White, P. D.;Oxford University Press, New York; Chap. 9, pp 215-228). In this methodsegments of the peptide usually in side-chain protected form, areprepared separately by either solution phase synthesis or solid phasesynthesis or a combination of the two methods. The choice of segments iscrucial and is made using a division strategy that can provide amanageable number of segments whose C-terminal residues and N-terminalresidues are projected to provide the cleanest coupling in peptidesynthesis. The C-terminal residues of the best segments are eitherdevoid of chiral alpha carbons (glycine or other moieties achiral at thecarbon ∝ to the carboxyl group to be activated in the coupling step) orare compromised of amino acids whose propensity to racemization duringactivation and coupling is lowest of the possible choices. The choice ofN-terminal amino acid for each segment is based on the ease of couplingof an activated acyl intermediate to the amino group. Once the divisionstrategy is selected the method of coupling of each of the segments ischosen based on the synthetic accessibility of the requiredintermediates and the relative ease of manipulation and purification ofthe resulting products (if needed). The segments are then coupledtogether, both in solution, or one on solid phase and the other insolution to prepare the final structure in fully or partially protectedform.

The protected target compound is then subjected to removal of protectinggroups, purified and isolated to give the final desired compound.Advantages of the segment coupling approach are that each segment can bepurified separately, allowing the removal of side products such asdeletion sequences resulting from incomplete couplings or those derivedfrom reactions such as side-chain amide dehydration during couplingsteps, or internal cyclization of side-chains (such as that of Gln) tothe alpha amino group during deprotection of Fmoc groups. Such sideproducts would all be present in the final product of a conventionalresin-based ‘straight through’ peptide chain assembly whereas removal ofthese materials can be performed, if needed, at many stages in a segmentcoupling strategy. Another important advantage of the segment couplingstrategy is that different solvents, reagents and conditions can beapplied to optimize the synthesis of each of the segments to high purityand yield resulting in improved purity and yield of the final product.Other advantages realized are decreased consumption of reagents andlower costs.

EXAMPLES

The following examples are provided as examples of different methodswhich can be used to prepare various compounds of the present invention.Within each example, there are compounds identified in single boldcapital letter (e.g., A, B, C), which correlate to the same labeledcorresponding compounds in the drawings identified.

General Experimental

A. Definitions of Additional Abbreviations Used

The following common abbreviations are used throughout thisspecification:

-   -   1,1-dimethylethoxycarbonyl (Boc or Boc);    -   9-fluorenylmethyloxycarbonyl (Fmoc);    -   allyloxycarbonyl (Aloc);    -   1-hydroxybenozotriazole (HOBt or HOBT);    -   N,N′-diisopropylcarbodiimide (DIC);    -   N-methylpyrrolidinone (NMP);    -   acetic anhydride (Ac₂O);    -   (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl        (iv-Dde);    -   trifluoroacetic acid (TFA);    -   Reagent B (TFA:H₂O:phenol:triisopropylsilane, 88:5:5:2);        diisopropylethylamine (DIEA);    -   O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate (HBTU);    -   O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorphosphate (HATU);    -   N-hydroxysuccinimide (NHS);    -   solid phase peptide synthesis (SPPS);    -   dimethylsulfoxide (DMSO);    -   dichloromethane (DCM);    -   dimethylformamide (DMF);    -   dimethylacetamide (DMA);    -   1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid        (DOTA);    -   Triisopropylsilane (TIPS);    -   1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid        (DOTA)    -   (1R)-1-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]ethane-1,2-dicarboxylic        acid (CMDOTA);    -   fetal bovine serum (FBS);    -   human serum albumin (HSA);    -   human prostate cancer cell line (PC3);    -   isobutylchloroformate (IBCF);    -   tributyl amine (TBA);    -   radiochemical purity (RCP); and    -   high performance liquid chromatography (HPLC).

B. Materials

The Fmoc-protected amino acids used were purchased from Nova-Biochem(San Diego, Calif., USA), Advanced Chem Tech (Louisville, Ky., USA),Chem-Impex International (Wood Dale Ill., USA), and Multiple PeptideSystems (San Diego, Calif., USA). Other chemicals, reagents andadsorbents required for the syntheses were procured from AldrichChemical Co. (Milwaukee, Wis., USA) and VWR Scientific Products(Bridgeport, N.J., USA). Solvents for peptide synthesis were obtainedfrom Pharmco Co. (Brookfield Conn., USA). Columns for HPLC analysis andpurification were obtained from Waters Co. (Milford, Mass., USA).Experimental details are given below for those that were notcommercially available.

C. Instrumentation for Peptide Synthesis

Peptides were prepared using an Advanced ChemTech 496Ω MOS synthesizer,an Advanced ChemTech 357 FBS synthesizer and/or by manual peptidesynthesis. However the protocols for iterative deprotection and chainextension employed were the same for all.

D. Automated Synthesis with the Symphony Instrument (Made by Rainin)

The synthesis was run with Symphony Software (Version 3) supplied byProtein Technologies Inc. Novagel TGR resin, with a substitution of 0.25mmol/g, was used, and each well contained 0.2 g of the resin (50 μmol).The amino acids were dissolved in NMP and the concentration was 0.25M. A0.25M solution of HBTU and N-Methylmorpholine in DMF was prepared andused for the coupling. All the couplings were carried out for 2.0 h. Thecleavage was done outside the machine by transferring the resin toanother reaction vessel and using Reagent B as in the manual synthesis

E. Instrumentation Employed for Analysis and Purification

Analytical HPLC was performed using a Shimadzu-LC-10A dual pump gradientanalytical LC system employing Shimadzu-ClassVP software version 4.1 forsystem control, data acquisition, and post run processing. Mass spectrawere acquired on a Hewlett-Packard Series 1100 MSD mass spectrometerinterfaced with a Hewlett-Packard Series 1100 dual pump gradient HPLCsystem fitted with an Agilent Technologies 1100 series autosamplerfitted for either direct flow injection or injection onto a WatersAssociates XTerra MS C18 column (4.6 mm×50 mm, 5μ particle, 120 Å pore).The instrument was driven by a HP Kayak workstation using ‘MSD Anyone’software for sample submission and HP Chemstation software forinstrument control and data acquisition. In most cases the samples wereintroduced via direct injection using a 5 μL injection of samplesolution at a concentration of 1 mg/mL and analyzed using positive ionelectrospray to obtain m/e and m/z (multiply charged) ions forconfirmation of structure. ¹H-NMR spectra were obtained on a VarianInnova spectrometer at 500 MHz. ¹³C-NMR spectra were obtained on thesame instrument at 125.73 MHz. Generally the residual ¹H absorption, orin the case of ¹³C-NMR, the ¹³C absorption of the solvent employed, wasused as an internal reference; in other cases tetramethylsilane (δ=0.00ppm) was employed. Resonance values are given in δ units. Micro analysisdata was obtained from Quantitative Technologies Inc., Whitehouse N.J.Preparative HPLC was performed on a Shimadzu-LC-8A dual pump gradientpreparative HPLC system employing Shimadzu-ClassVP software version 4.3for system control, data acquisition, fraction collection and post runprocessing.

F. General Procedures for Peptide Synthesis

Rink Amide-Novagel HL resin (0.6 mmol/g) was used as the solid support.

G. Coupling Procedure

In a typical experiment, the first amino acid was loaded onto 0.1 g ofthe resin (0.06 mmol). The appropriate Fmoc-amino acid in NMP (0.25Msolution; 0.960 mL was added to the resin followed byN-hydroxybenzotriazole (0.5M in NMP; 0.48 mL) and the reaction block (inthe case of automated peptide synthesis) or individual reaction vessel(in the case of manual peptide synthesis) was shaken for about 2 min. Tothe above mixture, diisopropylcarbodiimide (0.5M in NMP; 0.48 mL) wasadded and the reaction mixture was shaken for 4 h at ambienttemperature. Then the reaction block or the individual reaction vesselwas purged of reactants by application of a positive pressure of drynitrogen.

H. Washing Procedure

Each well of the reaction block was filled with 1.2 mL of NMP and theblock was shaken for 5 min. The solution was drained under positivepressure of nitrogen. This procedure was repeated three times. The sameprocedure was used, with an appropriate volume of NMP, in the case ofmanual synthesis using individual vessels.

I. Removal of Fmoc Protecting Group

The resin bearing the Fmoc-protected amino acid was treated with 1.5 mLof 20% piperidine in DMF (v/v) and the reaction block or individualmanual synthesis vessel was shaken for 15 min. The solution was drainedfrom the resin. This procedure was repeated once and the resin waswashed employing the washing procedure described above.

J. Final Coupling of Ligand (DOTA and CMDOTA)

The N-terminal amino group of the resin bound peptide linker constructwas deblocked and the resin was washed. A 0.25M solution of the desiredligand and HBTU in NMP was made, and was treated with a two-foldequivalency of DIEA. The resulting solution of activated ligand wasadded to the resin (1.972 mL; 0.48 mmol) and the reaction mixture wasshaken at ambient temperature for 24-30 h. The solution was drained andthe resin was washed. The final wash of the resin was conducted with 1.5mL dichloromethane (3×).

K. Deprotection and Purification of the Final Peptide

A solution of Reagent B (2 mL; 88:5:5:2—TFA:phenol:water:TIPS) was addedto the resin and the reaction block or individual vessel was shaken for4.5 h at ambient temperature. The resulting solution containing thedeprotected peptide was drained into a vial. This procedure was repeatedtwo more times with 1 mL of Reagent B. The combined filtrate wasconcentrated under reduced pressure using a Genevac HT-12 series IIcentrifugal concentrator. The residue in each vial was then trituratedwith 2 mL of Et₂O and the supernatant was decanted. This procedure wasrepeated twice to provide the peptides as colorless solids. The crudepeptides were dissolved in water/acetonitrile and purified using eithera Waters XTerra MS C18 preparative HPLC column (50 mm×19 mm, 5 micronparticle size, 120 Å pore size) or a Waters-YMC C18 ODS column (250mm×30 mm i.d., 10 micron particle size. 120 Å pore size). Theproduct-containing fractions were collected and analyzed by HPLC. Thefractions with >95% purity were pooled and the peptides isolated bylyophilization.

Conditions for Preparative HPLC (Waters XTerra Column):

-   -   Elution rate: 50 mL/min    -   Detection: UV, λ=220 nm    -   Eluent A: 0.1% aq. TFA; Eluent B: Acetonitrile (0.1% TFA).        -   Conditions for HPLC Analysis:    -   Column: Waters XTerra (Waters Co.; 4.6×50 mm; MS C18; 5 micron        particle, 120 Å pore).    -   Elution rate: 3 mL/min; Detection: UV, λ=220 nm.    -   Eluent A: 0.1% aq. TFA; Eluent B: Acetonitrile (0.1% TFA).

Example 1 FIGS. 1A-B Synthesis of L62

Summary: As shown in FIGS. 1A-B, L62 was prepared using the followingsteps: Hydrolysis of (3β,5β)-3-aminocholan-24-oic acid methyl ester Awith NaOH gave the corresponding acid B, which was then reacted withFmoc-Cl to give intermediate C. Rink amide resin functionalised with theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ IDNO:1]) was sequentially reacted with C, Fmoc-glycine and DOTAtri-t-butyl ester. After cleavage and deprotection with Reagent B thecrude was purified by preparative HPLC to give L62. Overall yield: 2.5%.More details are provided below:

A. Rink Amide Resin Functionalised with Bombesin[7-14], (A)

In a solid phase peptide synthesis vessel (see enclosure No. 1)Fmoc-aminoacid (24 mmol), N-hydroxybenzotriazole (HOBt) (3.67 g; 24mmol), and N,N′-diisopropylcarbodiimide (DIC) (3.75 mL; 24 mmol) wereadded sequentially to a suspension of Rink amide NovaGel™ resin (10 g;6.0 mmol) A in DMF (45 mL). The mixture was shaken for 3 h at roomtemperature using a bench top shaker, then the solution was emptied andthe resin was washed with DMF (5×45 mL). The resin was shaken with 25%piperidine in DMF (45 mL) for 4 min, the solution was emptied and fresh25% piperidine in DMF (45 mL) was added. The suspension was shaken for10 min, then the solution was emptied and the resin was washed with DMF(5×45 mL).

This procedure was applied sequentially for the following amino acids:N-α-Fmoc-L-methionine, N-α-Fmoc-L-leucine,N-α-Fmoc-N^(im)-trityl-L-histidine, N-α-Fmoc-glycine, N-α-Fmoc-L-valine,N-α-Fmoc-L-alanine, N-α-Fmoc-N^(in)-Boc-L-tryptophan.

In the last coupling reaction N-α-Fmoc-N-γ-trityl-L-glutamine (14.6 g;24 mmol), HOBt (3.67 g; 24 mmol), and DIC (3.75 mL; 24 mmol) were addedto the resin in DMF (45 mL). The mixture was shaken for 3 h at roomtemperature, the solution was emptied and the resin was washed with DMF(5×45 mL), CH₂Cl₂ (5×45 mL) and vacuum dried.

B. Preparation of Intermediates B and C (FIG. 1A) 1. Synthesis of(3β,5β)-3-Aminocholan-24-oic acid (B)

A 1 M solution of NaOH (16.6 mL; 16.6 mmol) was added dropwise to asolution of (3β,5β)-3-aminocholan-24-oic acid methyl ester (5.0 g; 12.8mmol) in MeOH (65 mL) at 45° C. After 3 h stirring at 45° C., themixture was concentrated to 25 mL and H₂O (40 mL) and 1 M HCl (22 mL)were added. The precipitated solid was filtered, washed with H₂O (2×50mL) and vacuum dried to give B as a white solid (5.0 g; 13.3 mmol).Yield 80%.

2. Synthesis of (3β,5β)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oicacid (C)

A solution of 9-fluorenylmethoxycarbonyl chloride (0.76 g; 2.93 mmol) in1,4-dioxane (9 mL) was added dropwise to a suspension of(3β,5β)-3-aminocholan-24-oic acid B (1.0 g; 2.66 mmol) in 10% aq. Na₂CO₃(16 mL) and 1,4-dioxane (9 mL) stirred at 0° C. After 6 h stirring atroom temperature H₂O (90 mL) was added, the aqueous phase washed withEt₂O (2×90 mL) and then 2 M HCl (15 mL) was added (final pH: 1.5). Theaqueous phase was extracted with EtOAc (2×100 mL), the organic phasedried over Na₂SO₄ and evaporated. The crude was purified by flashchromatography to give C as a white solid (1.2 g; 2.0 mmol). Yield 69%.

C. Synthesis of L62(N-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 1B)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas shaken for 20 min then the solution was emptied and the resin washedwith DMA (5×7 mL). (3β,5β)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oicacid C (0.72 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2mmol), N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7mL) were added to the resin, the mixture shaken for 24 h at roomtemperature, and the solution was emptied and the resin washed with DMA(5×7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL)for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL)was added and the mixture shaken for another 20 min. The solution wasemptied and the resin washed with DMA (5×7 mL). N-α-Fmoc-glycine (0.79g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol) and DMA(7 mL) were added to the resin. The mixture was shaken for 3 h at roomtemperature, the solution was emptied and the resin washed with DMA (5×7mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) wasadded and the mixture shaken for another 20 min. The solution wasemptied and the resin washed with DMA (5×7 mL) followed by addition of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol)and DMA (7 mL) to the resin. The mixture was shaken for 24 h at roomtemperature, the solution was emptied and the resin washed with DMA (5×7mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken in a flaskwith Reagent B (25 mL) for 4.5 h. The resin was filtered and thesolution was evaporated under reduced pressure to afford an oily crudewhich was triturated with Et₂O (20 mL) gave a precipitate. Theprecipitate was collected by centrifugation and washed with Et₂O (3×20mL), then analysed by HPLC and purified by preparative HPLC. Thefractions containing the product were lyophilised to give L62 (6.6 mg;3.8×10⁻³ mmol) as a white solid. Yield 4.5%.

Example II FIGS. 2A-F Synthesis of L70, L73, L74, L115 and L116

Summary: The products were obtained by coupling of the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) (withappropriate side chain protection) on the Rink amide resin withdifferent linkers, followed by functionalization with DOTA tri-t-butylester. After cleavage and deprotection with Reagent B the final productswere purified by preparative HPLC. Overall yields 3-9%.

A. Synthesis of L70 (FIG. 2A):

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min then the solution was emptied and the resinwashed with DMA (5×7 mL). Fmoc-4-aminobenzoic acid (0.43 g; 1.2 mmol),HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) wereadded to the resin, the mixture shaken for 3 h at room temperature, thesolution was emptied and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution was emptied, fresh 50% morpholine in DMA (7 mL) was added andthe mixture was shaken for 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). Fmoc-glycine (0.36 g; 1.2 mmol) HATU(0.46 g; 1.2 mmol) and DIEA (0.40 mL; 2.4 mmol) were stirred for 15 minin DMA (7 mL) then the solution was added to the resin, the mixtureshaken for 2 h at room temperature, the solution was emptied and theresin washed with DMA (5×7 mL). The resin was then shaken with 50%morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50%morpholine in DMA (7 mL) was added and the mixture shaken for 20 min.The solution was emptied and the resin washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol)and DMA (7 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied and the resin washed withDMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken ina flask with Reagent B (25 mL) for 4 h. The resin was filtered and thefiltrate solution was evaporated under reduced pressure to afford anoily crude that was triturated with Et₂O (5 mL). The precipitate wascollected by centrifugation and washed with Et₂O (5×5 mL), then analysedby HPLC and purified by preparative HPLC. The fractions containing theproduct were lyophilised to give L70 as a white fluffy solid (6.8 mg;0.005 mmol). Yield 3%.

B. Synthesis of L73, L115 and L116 (FIGS. 2B-2E):

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min then the solution was emptied and the resinwashed with DMA (5×7 mL). Fmoc-linker-OH (1.2 mmol), HOBt (0.18 g; 1.2mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin,the mixture was shaken for 3 h at room temperature, the solution wasemptied and the resin was washed with DMA (5×7 mL). The resin was shakenwith 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied,fresh 50% morpholine in DMA (7 mL) was added and the mixture was shakenfor 20 min. The solution was emptied and the resin washed with DMA (5×7mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol)and DMA (7 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied and the resin washed withDMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken ina flask with Reagent B (25 mL) for 4 h. The resin was filtered and thesolution was evaporated under reduced pressure to afford an oily crudethat was triturated with Et₂O (5 mL). The precipitate was collected bycentrifugation and washed with Et₂O (5×5 mL), then analysed by HPLC andpurified by preparative HPLC. The fractions containing the product werelyophilised.

C. Synthesis of L74 (FIG. 2F):

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min then the solution was emptied and the resin waswashed with DMA (5×7 mL). Fmoc-isonipecotic acid (0.42 g; 1.2 mmol),HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) wereadded to the resin, the mixture was shaken for 3 h at room temperature,the solution was emptied and the resin was washed with DMA (5×7 mL). Theresin was shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution was emptied, fresh 50% morpholine in DMA (7 mL) was added andthe mixture was shaken for 20 min. The solution was emptied and theresin was washed with DMA (5×7 mL). Fmoc-glycine (0.36 g; 1.2 mmol),HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) wereadded to the resin, the mixture was shaken for 3 h at room temperature,the solution was emptied and the resin washed with DMA (5×7 mL). Theresin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution was emptied, fresh 50% morpholine in DMA (7 mL) was added andthe mixture shaken for 20 minutes. The solution was emptied and theresin was washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol)and DMA (7 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied and the resin was washedwith DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin wasshaken in a flask with Reagent B (25 mL) for 4 h. The resin was filteredand the solution was evaporated under reduced pressure to afford an oilycrude that was triturated with Et₂O (5 mL). The precipitate wascollected by centrifugation and washed with Et₂O (5×5 mL), then analysedby HPLC and purified by HPPLC. The fractions containing the product werelyophilised to give L74 as a white fluffy solid (18.0 mg; 0.012 mmol).Yield 8%.

Example III FIGS. 3A-E Synthesis of L67

Summary: Hydrolysis of (3β,5β)-3-amino-12-oxocholan-24-oic acid methylester A with NaOH gave the corresponding acid B, which was then reactedwith Fmoc-Glycine to give intermediate C. Rink amide resinfunctionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂(BBN[7-14] [SEQ ID NO:1]) was sequentially reacted with C, and DOTAtri-t-butyl ester. After cleavage and deprotection with Reagent B thecrude was purified by preparative HPLC to give L67. Overall yield: 5.2%.

A. Synthesis (3β,5β)-3-Amino-12-oxocholan-24-oic acid, (B) (FIG. 3A)

A 1 M solution of NaOH (6.6 mL; 6.6 mmol) was added dropwise to asolution of (3β,5β)-3-amino-12-oxocholan-24-oic acid methyl ester A (2.1g; 5.1 mmol) in MeOH (15 mL) at 45° C. After 3 h stirring at 45° C., themixture was concentrated to 25 mL then H₂O (25 mL) and 1 M HCl (8 mL)were added. The precipitated solid was filtered, washed with H₂O (2×30mL) and vacuum dried to give B as a white solid (1.7 g; 4.4 mmol). Yield88%.

B. Synthesis of(3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxocholan-24-oicacid (C) (FIG. 3A)

Tributylamine (0.7 mL; 3.1 mmol) was added dropwise to a solution ofN-α-Fmoc-glycine (0.9 g; 3.1 mmol) in THF (25 mL) stirred at 0° C.Isobutyl chloroformate (0.4 mL; 3.1 mmol) was subsequently added and,after 10 min, a suspension of tributylamine (0.6 mL; 2.6 mmol) and(3β,5β)-3-amino-12-oxocholan-24-oic acid B (1.0 g; 2.6 mmol) in DMF (30mL) was added dropwise, over 1 h, into the cooled solution. The mixturewas allowed to warm up and after 6 h the solution was concentrated to 40mL, then H₂O (50 mL) and 1 N HCl (10 mL) were added (final pH: 1.5). Theprecipitated solid was filtered, washed with H₂O (2×50 mL), vacuum driedand purified by flash chromatography to give C as a white solid (1.1 g;1.7 mmol). Yield 66%.

C. Synthesis of L67(N-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-tryptotphyl-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 3B and FIG. 3E)

Resin D (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min then the solution was emptied and the resin waswashed with DMA (5×7 mL).(3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino]-12-oxocholan-24-oicacid C (0.80 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2mmol) and DMA (7 mL) were added to the resin, the mixture was shaken for24 h at room temperature, the solution was emptied and the resin waswashed with DMA (5×7 mL). The resin was shaken with 50% morpholine inDMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine inDMA (7 mL) was added and the mixture was shaken for 20 min. The solutionwas emptied and the resin was washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol)and DMA (7 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied and the resin was washedwith DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin wasshaken in a flask with Reagent B (25 mL) for 4.5 h. The resin wasfiltered and the solution was evaporated under reduced pressure toafford an oily crude that was triturated with Et₂O (20 mL).

Example IV FIGS. 4A-H Synthesis of L63 and L64

Summary: Hydrolysis of(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid methyl ester 1bwith NaOH gave the intermediate 2b, which was then reacted withFmoc-glycine to give 3b. Rink amide resin functionalised with theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ IDNO:1]) was reacted with 3b and then with DOTA tri-t-butyl ester. Aftercleavage and deprotection with Reagent B the crude was purified bypreparative HPLC to give L64. The same procedure was repeated startingfrom intermediate 2a, already available, to give L63. Overall yields: 9and 4%, respectively.

A. Synthesis of (3β,5β,7α,12α)-3-Amino-7,12-dihydroxycholan-24-oic acid,(2b) (FIG. 4A)

A 1 M solution of NaOH (130 mL; 0.13 mol) was added dropwise to asolution of (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acidmethyl ester 1b (42.1 g; 0.10 mol) in MeOH (300 mL) heated at 45° C.After 3 h stirring at 45° C., the mixture was concentrated to 150 mL andH₂O (350 mL) was added. After extraction with CH₂Cl₂ (2×100 mL) theaqueous solution was concentrated to 200 mL and 1 M HCl (150 mL) wasadded. The precipitated solid was filtered, washed with H₂O (2×100 mL)and vacuum dried to give 2b as a white solid (34.8 g; 0.08 mol). Yield80%.

B. Synthesis of(3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oicacid, (3a) (FIG. 4A)

Tributylamine (4.8 mL; 20.2 mmol) was added dropwise to a solution ofN-α-Fmoc-glycine (6.0 g; 20.2 mmol) in THF (120 mL) stirred at 0° C.Isobutyl chloroformate (2.6 mL; 20.2 mmol) was subsequently added and,after 10 min, a suspension of tributylamine (3.9 mL; 16.8 mmol) and(3β,5β,12α)-3-amino-12-hydroxycholan-24-oic acid 2a (6.6 g; 16.8 mmol)in DMF (120 mL) was added dropwise, over 1 h, into the cooled solution.The mixture was allowed to warm up and after 6 h the solution wasconcentrated to 150 mL, then H₂O (250 mL) and 1 N HCl (40 mL) were added(final pH: 1.5). The precipitated solid was filtered, washed with H₂O(2×100 mL), vacuum dried and purified by flash chromatography to give 3aas a white solid (3.5 g; 5.2 mmol). Yield 31%.

C. Synthesis of(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid, (3b) (FIG. 4A)

Tributylamine (3.2 mL; 13.5 mmol) was added dropwise to a solution ofN-α-Fmoc-glycine (4.0 g; 13.5 mmol) in THF (80 mL) stirred at 0° C.Isobutyl chloroformate (1.7 mL; 13.5 mmol) was subsequently added and,after 10 min, a suspension of tributylamine (2.6 mL; 11.2 mmol) and(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid 3a (4.5 g; 11.2mmol) in DMF (80 mL) was added dropwise, over 1 h, into the cooledsolution. The mixture was allowed to warm up and after 6 h the solutionwas concentrated to 120 mL, then H₂O (180 mL) and 1 N HCl (30 mL) wereadded (final pH: 1.5). The precipitated solid was filtered, washed withH₂O (2×100 mL), vacuum dried and purified by flash chromatography togive 3a as a white solid (1.9 g; 2.8 mmol). Yield 25%.

In an alternative method,(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid, (3b) can be prepared as follows:

N-Hydroxysuccinimide (1.70 g, 14.77 mmol) and DIC (1.87 g, 14.77 mmol)were added sequentially to a stirred solution of Fmoc-Gly-OH (4.0 g,13.45 mmol) in dichloromethane (15 mL); the resulting mixture wasstirred at room temperature for 4 h. The N,N′-diisopropylurea formed wasremoved by filtration and the solid was washed with ether (20 mL). Thevolatiles were removed and the solid Fmoc-Gly-succinimidyl ester formedwas washed with ether (3×20 mL). Fmoc-Gly-succinimidyl ester was thenredissolved in dry DMF (15 mL) and 3-aminodeoxycholic acid (5.21 g,12.78 mmol) was added to the clear solution. The reaction mixture wasstirred at room temperature for 4 h, water (200 mL) was added and theprecipitated solid was filtered, washed with water, dried and purifiedby silica gel chromatography (TLC (silica): (R_(f): 0.50, silica gel,CH₂Cl₂/CH₃OH, 9:1) (eluant: CH₂Cl₂/CH₃OH (9:1)) to give(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid as a colorless solid. Yield: 7.46 g (85%).

D. Synthesis of L63(N-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 4B)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min then the solution was emptied and the resinwashed with DMA (5×7 mL).(3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oicacid 3a (0.82 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2mmol) and DMA (7 mL) were added to the resin, the mixture was shaken for24 h at room temperature, the solution was emptied and the resin waswashed with DMA (5×7 mL). The resin was then shaken with 50% morpholinein DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholinein DMA (7 mL) was added and the mixture was shaken for 20 min. Thesolution was emptied and the resin washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol)and DMA (7 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied and the resin washed withDMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken ina flask with Reagent B (25 mL) for 4 h. The resin was filtered and thesolution was evaporated under reduced pressure to afford an oily crudethat after treatment with Et₂O (5 mL) gave a precipitate. Theprecipitate was collected by centrifugation and washed with Et₂O (5×5mL), then analysed and purified by HPLC. The fractions containing theproduct were lyophilised to give L63 as a white fluffy solid (12.8 mg;0.0073 mmol). Yield 9%.

E. Synthesis of L64(N-[(3β,5β,7α,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 4C)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min, the solution was emptied and the resin waswashed with DMA (5×7 mL).(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid 3b (0.81 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2mmol) and DMA (7 mL) were added to the resin, the mixture was shaken for24 h at room temperature, the solution was emptied and the resin waswashed with DMA (5×7 mL). The resin was shaken with 50% morpholine inDMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine inDMA (7 mL) was added and the mixture was shaken for 20 min. The solutionwas emptied and the resin was washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol)and DMA (7 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied and the resin washed withDMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken ina flask with Reagent B (25 mL) for 4 h. The resin was filtered and thesolution was evaporated under reduced pressure to afford an oily crudethat was triturated with Et₂O (5 mL). The precipitate was collected bycentrifugation and washed with Et₂O (5×5 mL). Then it was dissolved inH₂O (20 mL), and Na₂CO₃ (0.10 g; 0.70 mmol) was added; the resultingmixture was stirred 4 h at room temperature. This solution was purifiedby HPLC, the fractions containing the product lyophilised to give L64 asa white fluffy solid (3.6 mg; 0.0021 mmol). Yield 4%.

Example V FIGS. 5A-E Synthesis of L71 and L72

Summary: The products were obtained in two steps. The first step was thesolid phase synthesis of the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) (withappropriate side chain protecting groups) on the Rink amide resindiscussed supra. The second step was the coupling with different linkersfollowed by functionalization with DOTA tri-t-butyl ester. Aftercleavage and deprotection with Reagent B the final products werepurified by preparative HPLC. Overall yields 3-9%.

A. Bombesin [7-14] Functionalisation and Cleavage Procedure (FIGS. 5Aand 5D)

The resin B (0.5 g; 0.3 mmol) was shaken in a solid phase peptidesynthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, thesolution was emptied and fresh 50% morpholine in DMA (7 mL) was added.The suspension was stirred for 20 min then the solution was emptied andthe resin was washed with DMA (5×7 mL). The Fmoc-linker-OH (1.2 mmol),HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) wereadded to the resin. The mixture was shaken for 3 h at room temperature,the solution was emptied and the resin washed with DMA (5×7 mL). Theresin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution was emptied, fresh 50% morpholine in DMA (7 mL) was added andthe mixture was shaken for 20 min. The solution was emptied and theresin was washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl C (0.79 g; 1.2 mmol),HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4mmol) and DMA (7 mL) were added to the resin. The mixture was shaken for24 h at room temperature. The solution was emptied and the resin washedwith DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin wasshaken in a flask with Reagent B (25 mL) for 4 h. The resin was filteredand the filtrate was evaporated under reduced pressure to afford an oilycrude that was triturated with ether (5 mL). The precipitate wascollected by centrifugation and washed with ether (5×5 mL), thenanalyzed by analytical HPLC and purified by preparative HPLC. Thefractions containing the product were lyophilized.

B. Products 1. L71(4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

The product was obtained as a white fluffy solid (7.3 mg; 0.005 mmol).Yield 7.5%.

1. L72(Trans-4-[[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]cyclohexylcarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycil-L-histidyl-L-leucyl-L-methioninamide)

The product was obtained as a white fluffy solid (7.0 mg; 0.005 mmol).Yield 4.8%.

C.Trans-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]cyclohexanecarboxylicacid, (D) (FIG. 5E)

A solution of N-(9-fluorenylmethoxycarbonyloxy)succinimide (4.4 g; 14.0mmol) in 1,4-dioxane (40 mL) was added dropwise to a solution oftrans-4-(aminomethyl)cyclohexanecarboxylic acid (2.0 g; 12.7 mmol) in10% Na₂CO₃ (30 mL) cooled to 0° C. The mixture was then allowed to warmto ambient temperature and after 1 h stirring at room temperature wastreated with 1 N HCl (32 mL) until the final pH was 2. The resultingsolution was extracted with n-BuOH (100 mL); the volatiles were removedand the crude residue was purified by flash chromatography to give D asa white solid (1.6 g; 4.2 mmol). Yield 33%.

Example VI FIGS. 6A-F Synthesis of L75 and L76

Summary: The two products were obtained by coupling of the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) (A) on theRink amide resin with the two linkers E and H, followed byfunctionalization with DOTA tri-t-butyl ester. After cleavage anddeprotection with Reagent B the final products were purified bypreparative HPLC. Overall yields: 8.5% (L75) and 5.6% (L76).

A. 2-[(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic acid, (C)(FIG. 6A)

The product was synthesized following the procedure reported in theliterature (Bornstein, J; Drummon, P. E.; Bedell, S. F. Org. Synth.Coll. Vol. IV 1963, 810-812).

B. 2-(Aminomethyl)benzoic acid, (D) (FIG. 6A)

A 40% solution of methylamine (6.14 mL; 7.1 mmol) was added to2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic acid C (2 g;7.1 mmol) and then EtOH (30 mL) was added. After 5 minutes stirring atroom temperature the reaction mixture was heated at 50° C. After 2.5 h,the mixture was cooled and the solvent was evaporated under reducedpressure. The crude product was suspended in 50 mL of absolute ethanoland the suspension was stirred at room temperature for 1 h. The solidwas filtered and washed with EtOH to afford 2-(aminomethyl)benzoic acidD (0.87 g; 5.8 mmol). Yield 81%.

C. 2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid, (E)(FIG. 6A)

The product was synthesized following the procedure reported in theliterature (Sun, J-H.; Deneker, W. F. Synth. Commun. 1998, 28,4525-4530).

D. 4-(Aminomethyl)-3-nitrobenzoic acid, (G) (FIG. 6B)

4-(Bromomethyl)-3-nitrobenzoic acid (3.2 g; 12.3 mmol) was dissolved in8% NH₃ in EtOH (300 mL) and the resulting solution was stirred at roomtemperature. After 22 h the solution was evaporated and the residuesuspended in H₂O (70 mL). The suspension was stirred for 15 min andfiltered. The collected solid was suspended in H₂O (40 mL) and dissolvedby the addition of few drops of 25% aq. NH₄OH (pH. 12), then the pH ofthe solution was adjusted to 6 by addition of 6 N HCl. The precipitatedsolid was filtered, and washed sequentially with MeOH (3×5 mL), and Et₂O(10 mL) and was vacuum dried (1.3 kPa; P₂O₅) to give4-(aminomethyl)-3-nitrobenzoic acid as a pale brown solid (1.65 g; 8.4mmol). Yield 68%.

E. 4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoicacid, (H) (FIG. 6B)

4-(Aminomethyl)-3-nitrobenzoic acid G (0.8 g; 4 mmol) was dissolved in10% aq. Na₂CO₃ (25 mL) and 1,4-dioxane (10 mL) and the solution wascooled to 0° C. A solution of 9-fluorenylmethyl chloroformate (Fmoc-Cl)(1.06 g; 4 mmol) in 1,4-dioxane (10 mL) was added dropwise for 20 min.After 2 h at 0-5° C. and 1 h at 10° C. the reaction mixture was filteredand the solution was acidified to pH 5 by addition of 1 N HCl. Theprecipitate was filtered, washed with H₂O (2×2 mL) dried under vacuum(1.3 kPa; P₂O₅) to give4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acidas a white solid (1.6 g; 3.7 mmol). Yield 92%.

F. L75(N-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 6C)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min then the solution was emptied and the resinwashed with DMA (5×7 mL).2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid, E (0.45g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2 mmol),N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7 mL)were added to the resin, the mixture shaken for 24 h at roomtemperature, the solution was emptied and the resin was washed with DMA(5×7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL)for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL)was added and the mixture shaken for 20 min. The solution was emptiedand the resin washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (DOTA tri-t-butyl ester)(0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol),DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. Themixture was shaken for 24 h at room temperature, the solution wasemptied and the resin was washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) andvacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for4.5 h. The resin was filtered and the filtrate was evaporated underreduced pressure to afford an oily crude that after treatment with Et₂O(20 mL) gave a precipitate. The resulting precipitate was collected bycentrifugation and was washed with Et₂O (3×20 mL) to give L75 (190 mg;0.13 mmol) as a white solid. Yield 44%.

G. L76(N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 6D)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solution wasemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for 20 min then the solution was emptied and the resin waswashed with DMA (5×7 mL).4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acid,H (0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol)and DMA (7 mL) were added to the resin, the mixture was shaken for 24 hat room temperature, the solution was emptied and the resin was washedwith DMA (5×7 mL). The resin was then shaken with 50% morpholine in DMA(7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA(7 mL) was added and the mixture was shaken for 20 min. The solution wasemptied and the resin was washed with DMA (5×7 mL). DOTA tri-t-butylester (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin.The mixture was shaken for 24 h at room temperature, the solution wasemptied and the resin was washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) andvacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for4.5 h. The resin was filtered and the solution was evaporated underreduced pressure to afford an oily crude that was triturated with Et₂O(20 mL). The precipitate was collected by centrifugation and was washedwith Et₂O (3×20 mL) to give a solid (141 mg) which was analysed by HPLC.A 37 mg portion of the crude was purified by preparative HPLC. Thefractions containing the product were lyophilised to give L76 (10.8 mg;7.2×10⁻³ mmol) as a white solid. Yield 9%.

Example VII FIGS. 7A-C Synthesis of L124

Summary: 4-Cyanophenol A was reacted with ethyl bromoacetate and K₂CO₃in acetone to give the intermediate B, which was hydrolysed with NaOH tothe corresponding acid C. Successive hydrogenation of C with H₂ and PtO₂at 355 kPa in EtOH/CHCl₃ gave the corresponding aminoacid D, which wasdirectly protected with FmocOSu to give E. Rink amide resinfunctionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂(BBN[7-14] [SEQ ID NO:1]) was reacted with E and then with DOTAtri-t-butyl ester. After cleavage and deprotection with Reagent B thecrude was purified by preparative HPLC to give L124. Overall yield: 1.3%

A. Synthesis of (4-Cyanophenoxy)acetic acid ethyl ester, (B) (FIG. 7A)

The product was synthesized following the procedure reported in theliterature (Archimbault, P.; LeClerc, G.; Strosberg, A. D.;Pietri-Rouxel, F. PCT Int. Appl. WO 980005, 1998).

B. Synthesis of (4-Cyanophenoxy)acetic acid, (C) (FIG. 7A)

A 1 N solution of NaOH (7.6 mL; 7.6 mmol) was added dropwise to asolution of (4-cyanophenoxy)acetic acid ethyl ester B (1.55 g; 7.6 mmol)in MeOH (15 mL). After 1 h the solution was acidified with 1 N HCl (7.6mL; 7.6 mmol) and evaporated. The residue was taken up with water (20mL) and extracted with CHCl₃ (2×30 mL). The organic phases wereevaporated and the crude was purified by flash chromatography to give(4-cyanophenoxy)acetic acid C (0.97 g; 5.5 mmol) as a white solid. Yield72%.

C. Synthesis of[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid,(E) (FIG. 7A)

PtO₂ (150 mg) was added to a solution of (4-cyanophenoxy)acetic acid C(1.05 g; 5.9 mmol) in EtOH (147 mL) and CHCl₃ (3 mL). The suspension wasstirred 30 h under a hydrogen atmosphere (355 kPa; 20° C.). The mixturewas filtered through a Celite® pad and the solution evaporated undervacuum. The residue was purified by flash chromatography to give acid D(0.7 g) which was dissolved in H₂O (10 mL), MeCN (2 mL) and Et₃N (0.6mL) at 0° C., then a solution ofN-(9-fluorenylmethoxycarbonyloxy)succinimide (1.3 g; 3.9 mmol) in MeCN(22 mL) was added dropwise. After stirring 16 h at room temperature thereaction mixture was filtered and the volatiles were removed undervacuum. The residue was treated with 1 N HCl (10 mL) and theprecipitated solid was filtered and purified by flash chromatography togive [4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]aceticacid E (0.56 g; 1.4 mmol) as a white solid. Overall yield 24%.

D. Synthesis of L124(N-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 7B)

Resin A (480 mg; 0.29 mmol) was shaken in a solid phase peptidesynthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, thesolution was emptied and fresh 50% morpholine in DMA (7 mL) was added.The suspension was stirred for 20 min, the solution was emptied and theresin was washed with DMA (5×7 mL).[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid E(480 mg; 1.19 mmol), N-hydroxybenzotriazole (HOBt) (182 mg; 1.19 mmol),N,N′-diisopropylcarbodiimide (DIC) (185 μL; 1.19 mmol) and DMA (7 mL)were added to the resin, the mixture was shaken for 24 h at roomtemperature, the solution was emptied and the resin was washed with DMA(5×7 mL). The resin was then shaken with 50% morpholine in DMA (6 mL)for 10 min, the solution was emptied, fresh 50% morpholine in DMA (6 mL)was added and the mixture was shaken for 20 min. The solution wasemptied and the resin was washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (750 mg; 1.19 mmol), HOBt(182 mg; 1.19 mmol), DIEA (404 μL; 2.36 mmol), DIC (185 μL; 1.19 mmol)and DMA (6 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied, the resin was washed withDMA (2×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken ina flask with Reagent B (25 mL) for 4 h. The resin was filtered and thefiltrate was evaporated under reduced pressure to afford an oily crudethat was triturated with Et₂O (5 mL). The precipitate was collected bycentrifugation and washed with Et₂O (5×5 mL) to give a solid (148 mg)which was analysed by HPLC. A 65 mg portion of the crude was purified bypreparative HPLC. The fractions containing the product were lyophilisedto give L124 (FIG. 7C) as a white solid (15 mg; 0.01 mmol). Yield 7.9%.

Example VIII FIGS. 8A-C Synthesis of L125

Summary: 4-(Bromomethyl)-3-methoxybenzoic acid methyl ester A wasreacted with NaN₃ in DMF to give the intermediate azide B, which wasthen reduced with Ph₃P and H₂O to amine C. Hydrolysis of C with NaOHgave acid D, which was directly protected with FmocOSu to give E. Rinkamide resin functionalised with the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) (A) wasreacted with E and then with DOTA tri-t-butyl ester. After cleavage anddeprotection with Reagent B the crude was purified by preparative HPLCto give L125. Overall yield: 0.2%.

A. Synthesis of 4-(Azidomethyl)-3-methoxybenzoic acid methyl ester, (B)(FIG. 8A)

A solution of 4-(bromomethyl)-3-methoxybenzoic acid methyl ester (8 g;31 mmol) and NaN₃ (2 g; 31 mmol) in DMF (90 mL) was stirred overnight atroom temperature. The volatiles were removed under vacuum and the crudeproduct was dissolved in EtOAc (50 mL). The solution was washed withwater (2×50 mL) and dried. The volatiles were evaporated to provide4-(azidomethyl)-3-methoxybenzoic acid methyl ester (6.68 g; 30 mmol).Yield 97%.

B. 4-(Aminomethyl)-3-methoxybenzoic acid methyl ester, (C) (FIG. 8A)

Triphenylphosphine (6.06 g; 23 mmol) was added to a solution of(4-azidomethyl)-3-methoxybenzoic acid methyl ester B (5 g; 22 mmol) inTHF (50 mL): hydrogen evolution and formation of a white solid wasobserved. The mixture was stirred under nitrogen at room temperature.After 24 h more triphenylphosphine (0.6 g; 2.3 mmol) was added. After 24h the azide was consumed and H₂O (10 mL) was added. After 4 h the whitesolid disappeared. The mixture was heated at 45° C. for 3 h and wasstirred overnight at room temperature. The solution was evaporated todryness and the crude was purified by flash chromatography to give4-(aminomethyl)-3-methoxybenzoic acid methyl ester C (1.2 g; 6.1 mmol).Yield 28%.

C. 4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoicacid, (E) (FIG. 8A)

A 1 N solution of NaOH (6.15 mL; 6.14 mmol) was added dropwise to asolution of 4-(aminomethyl)-3-methoxybenzoic acid methyl ester C (1.2 g;6.14 mmol) in MeOH (25 mL) heated at 40° C. After stirring 8 h at 45° C.the solution was stirred over night at room temperature. A 1 N solutionof NaOH (0.6 mL; 0.6 mmol) was added and the mixture heated at 40° C.for 4 h. The solution was concentrated, acidified with 1 N HCl (8 mL; 8mmol), extracted with EtOAc (2×10 mL) then the aqueous layer wasconcentrated to 15 mL. This solution (pH 4.5) was cooled at 0° C. andEt₃N (936 μL; 6.75 mmol) was added (pH 11). A solution ofN-(9-fluorenylmethoxycarbonyloxy)succinimide (3.04 g; 9 mmol) in MeCN(30 mL) was added dropwise (final pH 9) and a white solid precipitated.After stirring 1 h at room temperature the solid was filtered, suspendedin 1N HCl (15 mL) and the suspension was stirred for 30 min. The solidwas filtered to provide4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acidE as a white solid (275 mg; 0.7 mmol).

The filtrate was evaporated under vacuum and the resulting white residuewas suspended in 1N HCl (20 mL) and stirred for 30 minutes. The solidwas filtered and purified by flash chromatography to give more acid E(198 mg; 0.5 mmol). Overall yield 20%.

D. L125(N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)(FIG. 8B)

Resin A (410 mg; 0.24 mmol) was shaken in a solid phase peptidesynthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, thesolution was emptied and fresh 50% morpholine in DMA (7 mL) was added.The suspension was stirred for 20 min then the solution was emptied andthe resin was washed with DMA (5×7 mL).4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acidE (398 mg; 0.98 mmol), HOBt (151 mg; 0.98 mmol), DIC (154 μL; 0.98 mmol)and DMA (6 mL) were added to the resin; the mixture was shaken for 24 hat room temperature, the solution was emptied and the resin was washedwith DMA (5×7 mL). The resin was then shaken with 50% morpholine in DMA(6 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA(6 mL) was added and the mixture was shaken for 20 min. The solution wasemptied and the resin washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (618 mg; 0.98 mmol), HOBt(151 mg; 0.98 mmol), DIC (154 μL; 0.98 mmol), DIEA (333 μL; 1.96 mmol)and DMA (6 mL) were added to the resin. The mixture was shaken for 24 hat room temperature, the solution was emptied and the resin was washedwith DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin wasshaken in a flask with Reagent B (25 mL) for 4 h. The resin was filteredand the solution was evaporated under reduced pressure to afford an oilycrude that was triturated with Et₂O (5 mL). The resulting precipitatewas collected by centrifugation, was washed with Et₂O (5×5 mL), wasanalysed by HPLC and purified by preparative HPLC. The fractionscontaining the product were lyophilised to give L125 (FIG. 8C) as awhite solid (15.8 mg; 0.011 mmol). Yield 4.4%.

Example IX FIGS. 9A-9D Synthesis of L146, L233, L234, and L235

Summary: The products were obtained in several steps starting from theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂(BBN[7-14]) (SEQ IDNO: 1) (A) on the Rink amide resin. After final cleavage anddeprotection with Reagent B the crudes were purified by preparative HPLCto give L146, L233, L234 and L235. Overall yields: 10%, 11%, 4.5%, 5.7%respectively.

A. 3-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminobenzoic acid,B (FIG. 9A)

A solution of 3-aminobenzoic acid (0.5 g; 3.8 mmol) andN-ethyldiisopropylamine (DIEA) (0.64 mL; 3.8 mmol) in THF (20 mL) wasadded dropwise to a solution of Fmoc-glycine chloride (1.2 g; 4.0 mmol)(3) in THF (10 mL) and CH₂Cl₂ (10 mL). After 24 h stirring at roomtemperature 1 M HCl (50 mL) was added (final pH: 1.5). The precipitatewas filtered, washed with H₂O (2×100 mL), vacuum dried and crystallisedfrom CHCl₃/CH₃OH (1:1) to give B as a white solid (0.7 g; 1.6 mmol).Yield 43%.

B.N-[3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L233 (FIG. 9D)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added.

The suspension was stirred for another 20 min then the solution wasemptied and the resin washed with DMA (5×7 mL).3-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminobenzoic acid, B(0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) andDMA (7 mL) were added to the resin, the mixture shaken for 6 h at roomtemperature, emptied and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). DOTA tri-t-butyl ester adduct with NaCl²(0.79 g; 1.2 mmol) (5), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin.The mixture was shaken for 24 h at room temperature, emptied and theresin washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. Theresin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resinwas filtered and the solution was evaporated under reduced pressure toafford an oily crude that after treatment with Et₂O (20 mL) gave aprecipitate. The precipitate was collected by centrifugation and washedwith Et₂O (3×20 mL) to give a solid (152 mg) which was analysed by HPLC.An amount of crude (50 mg) was purified by preparative HPLC. Thefractions containing the product were lyophilised to give L233 (17.0 mg;11.3×10⁻³ mmol) as a white solid. Yield 11%.

C.N-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L146 (FIG. 9D)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionfiltered and fresh 50% morpholine in DMA (7 mL) was added. Thesuspension was stirred for another 20 min then the solution was filteredand the resin washed with DMA (5×7 mL). Fmoc-4-aminophenylacetic acid(0.45 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) andDMA (7 mL) were added to the resin, the mixture shaken for 6 h at roomtemperature, filtered and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution filtered, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was filtered and theresin washed with DMA (5×7 mL). Fmoc-glycine (0.36 g; 1.2 mmol), HATU(0.46 g; 1.2 mmol) and DIEA (0.40 mL; 2.4 mmol) were stirred for 15 minin DMA (7 mL) then the solution was added to the resin, the mixtureshaken for 2 h at room temperature, filtered and the resin washed withDMA (5×7 mL). The resin was then shaken with 50% morpholine in DMA (7mL) for 10 min, the solution filtered, fresh 50% morpholine in DMA (7mL) was added and the mixture shaken for another 20 min. The solutionwas filtered and the resin washed with DMA (5×7 mL). DOTA tri-t-butylester adduct with NaCl (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC(0.19 mL; 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were addedto the resin. The mixture was shaken for 24 h at room temperature,filtered and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) andvacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for4.5 h. The resin was filtered and the solution was evaporated underreduced pressure to afford an oily crude that after treatment with Et₂O(20 mL) gave a precipitate. The precipitate was collected bycentrifugation and washed with Et₂O (3×20 mL) to give a solid (203 mg)which was analysed by HPLC. An amount of crude (50 mg) was purified bypreparative HPLC. The fractions containing the product were lyophilisedto give L146 (11.2 mg; 7.4×10-3 mmol) as a white solid. Yield 10%.

D. 6-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminonaphthoicacid, C (FIG. 9B)

A solution of 6-aminonaphthoic acid (500 mg; 2.41 mmol); and DIEA (410μL 2.41 mmol) in THF (20 mL) was added dropwise to a solution ofFmoc-glycine chloride (760 mg; 2.41 mmol) in CH₂Cl₂/THF 1:1 (10 mL) andstirred at room temperature. After 24 h the solvent was evaporated undervacuum. The residue was taken up with 0.5 N HCl (50 mL) and stirred for1 h. The white solid precipitated was filtered and dried. The whitesolid was suspended in methanol (30 mL) and boiled for 5 min, then wasfiltered to give product C (690 mg; 1.48 mmol). Yield 62%.

E.N-[6-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]naphthoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L234

Resin A (500 mg; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL).6-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminonaphthoic acid C(560 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 μL; 1.2 mmol) andDMA (7 mL) were added to the resin, the mixture shaken for 6 h at roomtemperature, emptied and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (6 L) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). DOTA tri-t-butyl ester adduct with NaCl(757 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 μL; 1.2 mmol), andDIEA (537 μL; 2.4 mmol) and DMA (7 mL) were added to the resin. Themixture was shaken in a flask, emptied and the resin washed with DMA(2×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken in aflask with Reagent B (25 mL) for 4.5 h. The resin was filtrated and thesolution was evaporated under reduced pressure to afford an oil crudethat after treatment with Et₂O (20 mL) gave a precipitate. Theprecipitate was collected by centrifugation and washed with Et₂O (3×20mL) to give a solid (144 mg) which was analysed by HPLC. An amount ofcrude (54 mg) was purified by preparative HPLC. The fractions containingthe product were lyophilised to give L234 (8 mg; 5.1×10⁻³ mmol) as awhite solid. Yield 4.5%.

F.4-[[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]methylamino]benzoicacid, D (FIG. 9C)

A solution of 4-N-methylaminonaphthoic acid (500 mg; 3.3 mmol) and DIEA(562 μL 3.3 mmol) in THF (20 mL) was added to a solution of Fmoc-glycinechloride (1.04 g; 3.3 mmol) in CH₂Cl₂/THF 1:1 (10 mL) and stirred atroom temperature. After 24 h the solvent was evaporated under vacuum.The residue was taken up with 0.5 N HCl (30 mL) and was stirred for 3 hat 0° C. The white solid precipitated was filtered and dried. The crudewas purified by flash chromatography to give Compound D (350 mg; 0.81mmol). Yield 25%.

G.N-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]methylamino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L235 (FIG. 9D)

Resin A (500 mg; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL).4-[[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]-N-methyl]amino-benzoicacid D (510 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 μL; 1.2mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 6 hat room temperature, emptied and the resin washed with DMA (5×7 mL). Theresin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). DOTA tri-t-butyl ester adduct with NaCl(757 mg; 1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 μL; 1.2 mmol), andDIEA (537 μL; 2.4 mmol) and DMA (7 mL) were added to the resin. Themixture was shaken in a flask, emptied and the resin washed with DMA(2×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken in aflask with Reagent B (25 mL) for 4.5 h. The resin was filtrated and thesolution was evaporated under reduced pressure to afford an oil crudethat after treatment with Et₂O (20 mL) gave a precipitate.

The precipitate was collected by centrifugation and washed with Et₂O(3×20 mL) to give a solid (126 mg) which was analysed by HPLC. An amountof crude (53 mg) was purified by preparative HPLC. The fractionscontaining the product were lyophilised to give L235 (11 mg; 7.2×10⁻³mmol) as a white solid. Yield 5.7%.

Example X FIGS. 10A-B Synthesis of L237

Summary: 1-Formyl-1,4,7,10-tetraazacyclododecane (A) was selectivelyprotected with benzyl chloroformate at pH 3 to give B, which wasalkylated with t-butyl bromoacetate and deformylated with hydroxylaminehydrochloride to give D. Reaction with P(OtBu)₃ and paraformaldehydegave E, which was deprotected by hydrogenation and alkylated with benzylbromoacetate to give G, which was finally hydrogenated to H. Rink amideresin functionalized with the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14]) (A) was sequentiallyreacted with Fmoc-4-aminobenzoic acid, Fmoc-glycine and H. Aftercleavage and deprotection with Reagent B the crude was purified bypreparative HPLC to give L237. Overall yield 0.21%.

A. 7-Formyl-1,4,7,10-tetraazacyclododecane-1-carboxylic acidphenylmethyl ester dihydrochloride, B (FIG. 10A)

1-Formyl-1,4,7,10-tetraazacyclododecane A (14 g; 69.9 mmol) wasdissolved in H₂O (100 mL) and 12 N HCl (11 mL) was added until pH 3 then1,4-dioxane (220 mL) was added. A solution of benzyl chloroformate (13.8g; 77 mmol) in 1,4-dioxane (15 mL) was slowly added dropwise in 3.5 h,constantly maintaining the reaction mixture at pH 3 by continuousaddition of 2 N NaOH (68.4 mL) with a pHstat apparatus. At the end ofthe addition the reaction was stirred for 1 h then washed with n-hexane(4×100 mL) and ^(i)Pr₂O (4×100 mL). The aqueous phase was brought to pH13 by addition of 10 N NaOH (6.1 mL) and extracted with CHCl₃ (4×100mL). The organic phase was washed with brine (100 mL), dried (Na₂SO₄),filtered and evaporated. The oily residue was dissolved in acetone (200mL) and 6 N HCl (26 mL) was added. The solid precipitated was filtered,washed with acetone (2×50 mL) and dried under vacuum to give compound B(23.6 g; 58 mmol) as a white crystalline solid. Yield 83%.

B. 4-(Phenylmethoxy)carbonyl-1,4,7,10-tetraazacyclododecane-1,7-diaceticacid bis(1,1-dimethylethyl) ester, D (FIG. 10A)

A solution of B (14.4 g; 35.3 mmol) in H₂O (450 mL) and 1 N NaOH (74 mL;74 mmol) was stirred for 20 min then extracted with CHCl₃ (4×200 mL).The organic layer was evaporated to obtain an oily residue (12.3 g)which was dissolved in CH₃CN (180 mL) and N-ethyldiisopropylamine (DIEA)(15 mL; 88.25 mmol). A solution of t-butyl bromoacetate (16.8 g; 86.1mmol) in CH₃CN (15 mL) was added dropwise to the previous solution in2.5 h. After 20 h at room temperature the solvent was evaporated and theoily residue was dissolved in CHCl₃ (150 mL) and washed with H₂O (5×100mL). The organic layer was dried (Na₂SO₄), filtered and evaporated todryness to give C as a yellow oil. Crude C (22 g) was dissolved in EtOH(250 mL), NH₂OH.HCl (2.93 g; 42.2 mmol) was added and the solutionheated to reflux. After 48 h the solvent was evaporated and the residuedissolved in CH₂Cl₂ (250 mL), washed with H₂O (3×250 mL) then with brine(3×250 mL). The organic layer was dried (Na₂SO₄), filtered andevaporated. The oily residue (18.85 g) was purified by flashchromatography. The fractions containing the product were collected andevaporated to obtain a glassy white solid (17.62 g) which was dissolvedin H₂O (600 mL) and 1 N NaOH (90 mL; 90 mmol) and extracted with CHCl₃(3×250 ml). The organic layer was dried (Na₂SO₄) and evaporated todryness to give D (16.6 g; 31 mmol) as an oil. Yield 88%.

C.4-(Phenylmethoxy)carbonyl-10-[[bis(1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-tetraazacyclododecane-1,7-diaceticacid bis(1,1-dimethylethyl) ester, E (FIG. 10A)

A mixture of Compound D (13.87 g; 26 mmol), P(OtBu)₃ (7.6 g; 28.6 mmol)(10) and paraformaldeyde (0.9 g; 30 mmol) was heated at 60° C. After 16h more P(OtBu)₃ (1 g; 3.76 mmol) and paraformaldeyde (0.1 g; 3.33 mmol)were added. The reaction was heated at 60° C. for another 20 h then at80° C. for 8 h under vacuum to eliminate the volatile impurities. Thecrude was purified by flash chromatography to give E (9.33 g; 8 mmol) asan oil. Yield 31%.

D.7-[[Bis(1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triaceticacid 1-phenylmethyl 4,10-bis(1,1-dimethylethyl) ester, G (FIG. 10A)

To the solution of E (6.5 g; 5.53 mmol) in CH₃OH (160 mL) 5% Pd/C (1 g;0.52 mmol) was added and the mixture was stirred under hydrogenatmosphere at room temperature. After 4 h (consumed H₂ 165 mL; 6.7 mmol)the mixture was filtered through a Millipore® filter (FT 0.45 μm) andthe solution evaporated under reduced pressure. The crude (5.9 g) waspurified by flash chromatography to give F (4.2 g) as an oil. Benzylbromoacetate (1.9 g; 8.3 mmol) dissolved in CH₃CN (8 mL) was addeddropwise in 1 h to a solution of F (4.2 g) in CH₃CN (40 mL) and DIEA(1.5 mL; 8.72 mmol). After 36 h at room temperature the solvent wasevaporated and the residue (5.76 g) dissolved in CHCl₃ (100 mL), washedwith H₂O (2×100 mL) then with brine (2×70 mL). The organic layer wasdried (Na₂SO₄), filtered and evaporated. The crude (5.5 g) was purifiedtwice by flash chromatography, the fractions were collected andevaporated to dryness to afford G (1.12 g; 1.48 mmol) as an oil. Yield27%.

E.7-[[Bis(1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triaceticacid 4,10-bis(1,1-dimethylethyl) ester, H (FIG. 10A)

5% Pd/C (0.2 g; 0.087 mmol) was added to a solution of G (1.12 g; 1.48mmol) in CH₃OH (27 mL) and the mixture was stirred under hydrogenatmosphere at room temperature. After 2 h (consumed H₂ 35 mL; 1.43 mmol)the mixture was filtered through a Millipore® filter (FT 0.45 μm) andthe solution evaporated to dryness to give H (0.94 g; 1.41 mmol) as apale yellow oil. Yield 97%.

F.N-[4-[[[[[4,10-Bis(carboxymethyl)-7-(dihydroxyphosphinyl)methyl-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucil-L-methioninamide,L237 (FIG. 10B)

Resin A (330 mg; 0.20 mmol) (17) was shaken in a solid phase peptidesynthesis vessel with 50% morpholine in DMA (5 mL) for 10 min, thesolution emptied and fresh 50% morpholine in DMA (5 mL) was added. Thesuspension was stirred for another 20 min then the solution was emptiedand the resin washed with DMA (5×5 mL). Fmoc-4-aminobenzoic acid (290mg; 0.80 mmol), HOBt (120 mg; 0.80 mmol), DIC (130 μL; 0.80 mmol) andDMA (5 mL) were added to the resin, the mixture shaken for 3 h at roomtemperature, emptied and the resin washed with DMA (5×5 mL). The resinwas then shaken with 50% morpholine in DMA (5 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (5 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×5 mL). Fmoc-glycine (240 mg; 0.8 mmol), HATU(310 mg; 0.8 mmol) and DIEA (260 μL; 1.6 mmol) were stirred for 15 minin DMA (5 mL) then the solution was added to the resin, the mixtureshaken for 2 h at room temperature, emptied and the resin washed withDMA (5×5 mL). The resin was then shaken with 50% morpholine in DMA (5mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (5 mL)was added and the mixture shaken for another 20 min. The solution wasemptied and the resin washed with DMA (5×5 mL). H (532 mg; 0.80 mmol),HOBt (120 mg; 0.80 mmol), DIC (130 μL; 0.80 mmol), and DIEA (260 μL; 1.6mmol) and DMA (5 mL) were added to the resin. The mixture was shaken ina flask for 40 h at room temperature, emptied and the resin washed withDMA (5×5 mL), CH₂Cl₂ (5×5 mL) and vacuum dried. The resin was shaken ina flask with Reagent B (25 mL) for 4 h. The resin was filtered and thesolution was evaporated under reduced pressure to afford an oily crudethat after treatment with Et₂O (20 mL) gave a precipitate. Theprecipitate was collected by centrifugation and washed with Et₂O (3×20mL) to give a solid (90 mg) which was analysed by HPLC. An amount ofcrude (50 mg) was purified by preparative HPLC. The fractions containingthe product were lyophilised to give L237 (6 mg; 3.9×10⁻³ mmol) as awhite solid. Yield 3.5%.

Example XI FIGS. 11A-B Synthesis of L238 and L239

Summary: The products were obtained in several steps starting from theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14]) (A) on theRink amide resin. After cleavage and deprotection with Reagent B thecrude was purified by preparative HPLC to give L238 and L239. Overallyields: 14 and 9%, respectively.

A.N,N-Dimethylglycyl-L-seryl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L238 (FIG. 11A)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL). Fmoc-4-aminobenzoic acid (0.43 g; 1.2mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL)were added to the resin, the mixture shaken for 3 h at room temperature,emptied and the resin washed with DMA (5×7 mL). The resin was thenshaken with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied, fresh 50% morpholine in DMA (7 mL) was added and the mixtureshaken for another 20 min. The solution was emptied and the resin washedwith DMA (5×7 mL). Fmoc-glycine (0.36 g; 1.2 mmol), HATU (0.46 g; 1.2mmol) and N-ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for15 min in DMA (7 mL) then the solution was added to the resin, themixture shaken for 2 h at room temperature, emptied and the resin washedwith DMA (5×7 mL). The resin was then shaken with 50% morpholine in DMA(7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7mL) was added and the mixture shaken for another 20 min. The solutionwas emptied and the resin washed with DMA (5×7 mL).N-α-Fmoc-S-acetamidomethyl-L-cysteine (0.50 g; 1.2 mmol), HOBt (0.18 g;1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to theresin, the mixture shaken for 3 h at room temperature, emptied and theresin washed with DMA (5×7 mL). The resin was then shaken with 50%morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50%morpholine in DMA (7 mL) was added and the mixture shaken for another 20min. The solution was emptied and the resin washed with DMA (5×7 mL).N-α-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol),DIC (0.19 mL: 1.2 mmol), and DMA (7 mL) were added to the resin, themixture was shaken for 3 h at room temperature, emptied and the resinwashed with DMA (5×7 mL). The resin was then shaken with 50% morpholinein DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine inDMA (7 mL) was added and the mixture shaken for another 20 min.

The solution was emptied and the resin washed with DMA (5×7 mL).N,N-Dimethylglycine (0.12 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) andN-ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min inDMA (7 mL) then the solution was added to the resin. The mixture wasshaken for 2 h at room temperature, emptied and the resin washed withDMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin was shaken ina flask with Reagent B (25 mL) for 4.5 h. The resin was filtered and thesolution was evaporated under reduced pressure to afford an oily crudethat after treatment with Et₂O (20 mL) gave a precipitate. Theprecipitate was collected by centrifugation and washed with Et₂O (3×20mL) to give a solid (169 mg) which was analysed by HPLC. An amount ofcrude (60 mg) was purified by preparative HPLC. The fractions containingthe product were lyophilised to give L238 (22.0 mg; 0.015 mmol) as awhite solid. Yield 14%.

B.N,N-Dimethylglycyl-L-seryl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-(3β,5β,7α,12α)-3-amino-7,12-dihydroxy-24-oxocholan-24-yl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L239 (FIG. 11B)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL).(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid B (0.82 g; 1.2 mmol) (7), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL;1.2 mmol) and DMA (7 mL) were added to the resin, the mixture shaken for24 h at room temperature, emptied and the resin washed with DMA (5×7mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was addedand the mixture shaken for another 20 min. The solution was emptied andthe resin washed with DMA (5×7 mL).N-α-Fmoc-S-acetamidomethyl-L-cysteine (0.50 g; 1.2 mmol), HOBt (0.18 g;1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to theresin, the mixture was shaken for 3 h at room temperature, emptied andthe resin washed with DMA (5×7 mL). The resin was then shaken with 50%morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50%morpholine in DMA (7 mL) was added and the mixture shaken for another 20min. The solution was emptied and the resin washed with DMA (5×7 mL).N-α-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol),DIC (0.19 mL: 1.2 mmol), and DMA (7 mL) were added to the resin, themixture was shaken for 3 h at room temperature, emptied and the resinwashed with DMA (5×7 mL). The resin was then shaken with 50% morpholinein DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine inDMA (7 mL) was added and the mixture shaken for another 20 min. Thesolution was emptied and the resin washed with DMA (5×7 mL).N,N-Dimethylglycine (0.12 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) andN-ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min inDMA (7 mL) then the solution was added to the resin.

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL).(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid B (0.82 g; 1.2 mmol) HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 24h at room temperature, emptied and the resin washed with DMA (5×7 mL).The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min,the solution emptied, fresh 50% morpholine in DMA (7 mL) was added andthe mixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). N-α-Fmoc-S-acetamidomethyl-L-cysteine(0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) andDMA (7 mL) were added to the resin, the mixture was shaken for 3 h atroom temperature, emptied and the resin washed with DMA (5×7 mL). Theresin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). N-α-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), and DMA (7 mL)were added to the resin, the mixture was shaken for 3 h at roomtemperature, emptied and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). N,N-Dimethylglycine (0.12 g; 1.2 mmol),HATU (0.46 g; 1.2 mmol) and N-ethyldiisopropylamine (0.40 mL; 2.4 mmol)were stirred for 15 min in DMA (7 mL) then the solution was added to theresin.

Example XII FIGS. 12A-F Synthesis of L240, L241, L242

Summary: The products were obtained in several steps starting from theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14]) (A) on theRink amide resin. After cleavage and deprotection with Reagent B thecrudes were purified by preparative HPLC to give L240, L241, and L242.Overall yields: 7.4, 3.2, 1.3% respectively.

A.4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methoxybenzoicacid A (FIG. 12A)

A solution of 4-amino-3-methoxybenzoic acid (1.0 g; 5.9 mmol); andN-ethyldiisopropylamine (1.02 mL 5.9 mmol) in THF (20 mL) was addeddropwise to a solution of Fmoc-glycylchloride (1.88 g; 5.9 mmol) inCH₂Cl₂/THF 1:1 (20 mL) and stirred at room temperature under N₂. After 3h the solvent was evaporated under vacuum. The residue was taken up with0.5 N HCl (50 mL), was stirred for 1 h at 0° C. then filtered and dried.The white solid was suspended in MeOH (30 mL) and stirred for 1 h, thenwas filtered and suspended in a solution of CHCl₃/hexane 1:4 (75 mL).The suspension was filtered to give compound A as a with solid (1.02 g;2.28 mmol). Yield 39%.

B.N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododec-1-yl]acetyl]glycyl]amino]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-1-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL240

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL).4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methoxybenzoicacid, A (0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 5 hat room temperature, emptied and the resin washed with DMA (5×7 mL). Theresin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), N-ethyldiisopropylamine(0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixturewas shaken for 24 h at room temperature, emptied and the resin washedwith DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin wasshaken in a flask with Reagent B (25 mL) for 4.5 h. The resin wasfiltered and the solution was evaporated under reduced pressure toafford an oil crude that after treatment with Et₂O (20 mL) gave aprecipitate. The precipitate was collected by centrifugation and washedwith Et₂O (5×20 mL) to give a solid (152 mg) which was analysed by HPLC.An amount of crude (52 mg) was purified by preparative HPLC. Thefractions containing the product were lyophilised to give L240 (12.0 mg;7.8×10⁻³ mmol) as a white solid. Yield 7.4%.

C. 4-amino-3-chlorobenzoic acid C (FIG. 12B)

1 N NaOH (11 mL; 11 mmol) was added to a solution of methyl4-amino-3-chlorobenzoate (2 g; 10.8 mmol) in MeOH (20 mL) at 45° C. Thereaction mixture was stirred for 5 h at 45° C. and overnight at roomtemperature. More 1N NaOH was added (5 mL; 5 mmol) and the reaction wasstirred at 45° C. for 2 h. After concentration of solvent was added 1NHCl (16 ml). The solid precipitate was filtered and dried to give4-amino-3-chlorobenzoic acid, C, as a with solid (1.75 g; 10.2 mmol).Yield 94.6%.

D.4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoicacid, D (FIG. 12B)

A solution of 4-amino-3-chlorobenzoic acid (1.5 g; 8.75 mmol) andN-ethyldiisopropylamine (1.46 mL 8.75 mmol) in THF (50 mL) was addeddropwise to a solution of Fmoc-glycylchloride (2.76 g; 8.75 mmol) inCH₂Cl₂/THF 1:1 (30 mL) and stirred at room temperature under N₂. After 3h the solvent was evaporated under vacuum. The residue was taken up with0.5N HCl (50 mL), filtered and dried.

The white solid was suspended in MeOH (30 mL) and stirred for 1 h, thenwas filtered and dried to give4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoicacid (2.95 g; 6.5 mmol). Yield 75%.

E.N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododec-1-yl]acetyl]glycyl]amino]3-chlorobenzoyl]L-glutaminyl-L-tryptophyl-1-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L241 (FIG. 12E)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL).4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoicacid, D (0.54 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2mmol) and DMA (7 mL) were added to the resin, the mixture shaken for 5 hat room temperature, emptied and the resin washed with DMA (5×7 mL).

The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min,the solution emptied, fresh 50% morpholine in DMA (7 mL) was added andthe mixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), N-ethyldiisopropylamine(0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixturewas shaken for 40 h at room temperature, emptied and the resin washedwith DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin wasshaken in a flask with Reagent B (25 mL) for 4.5 h. The resin wasfiltered and the solution was evaporated under reduced pressure toafford an oil crude that after treatment with Et₂O (20 mL) gave aprecipitate. The precipitate was collected by centrifugation and washedwith Et₂O (5×20 mL) to give a solid (151 mg) which was analysed by HPLC.An amount of crude (56 mg) was purified by preparative HPLC. Thefractions containing the product were lyophilised to give L241 (5.6 mg;3.6×10⁻³ mmol) as a white solid. Yield 3.2%.

F.4-[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methylbenzoicacid, E (FIG. 12C)

A solution of 4-amino-3-methylbenzoic acid (0.81 g; 5.35 mmol) andN-ethyldiisopropylamine (0.9 mL 5.35 mmol) in THF (30 mL) was addeddropwise to a solution of Fmoc-glycylchloride (1.69 g; 5.35 mmol) inCH₂Cl₂/THF 1:1 (20 mL) and stirred at room temperature under N₂. After 3h the solvent was evaporated under vacuum. The residue was taken up withHCl 0.5 N (50 mL) and was stirred for 3 h at 0° C. then was filtered anddried. The white solid was suspended in MeOH (50 mL) and stirred for 1h, then filtered and dried to give Compound E (1.69 g; 3.9 mmol). Yield73%.

G.N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]glycyl]amino]3-methylbenzoyl]L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL242 (FIG. 12F)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL).4-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-3-methylbenzoic acid, E(0.52 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) andDMA (7 mL) were added to the resin, the mixture shaken for 5 h at roomtemperature, emptied and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL).1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.76 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol), N-ethyldiisopropylamine(0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin.

The mixture was shaken for 40 h at room temperature, emptied and theresin washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. Theresin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resinwas filtered and the solution was evaporated under reduced pressure toafford an oil crude that after treatment with Et₂O (20 mL) gave aprecipitate. The precipitate was collected by centrifugation and washedwith Et₂O (5×20 mL) to give a solid (134 mg) which was analysed by HPLC.An amount of crude (103 mg) was purified by preparative HPLC. Thefractions containing the product were lyophilised to give L242 (4.5 mg;2.9×10⁻³ mmol) as a white solid. Yield 1.3%.

Example XIII FIGS. 13A-C Synthesis of L244

Summary: The product was obtained in several steps starting from theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14]) on the Rinkamide resin (A). The final coupling step with DOTA tri-t-butyl ester wasdone in solution phase after cleavage and deprotection with Reagent B ofLinker-BBN [7-14]. The crude was purified by preparative HPLC to giveL244. Overall yield: 0.4%.

A. N,N′-(Iminodi-2,1-ethanediyl)bis[2,2,2-trifluoroacetamide], A (FIG.13A)

Trifluoroacetic acid ethyl ester (50 g; 0.35 mol) was dropped into asolution of diethylenetriamine (18 g; 0.175 mol) in THF (180 mL) at 0°C. in 1 h. After 20 h at room temperature, the mixture was evaporated toan oily residue (54 g). The oil was crystallized from Et₂O (50 mL),filtered, washed with cooled Et₂O (2×30 mL) and dried to obtain A as awhite solid (46 g; 0.156 mol). Yield 89%.

B. 4-[N,N′-Bis[2-(trifluoroacetyl)aminoethyl]amino]-4-oxobutanoic acid,B (FIG. 13A)

Succinic anhydride (0.34 g; 3.4 mmol) was added in a solution of A (1 g;3.4 mmol) in THF (5 mL) at room temperature. After 28 h the crude wasconcentrated to residue (1.59 g), washed with EtOAc (2×10 mL) and 1 NHCl (2×15 mL). The organic layer was dried on Na₂SO₄, filtered andevaporated to give an oily residue (1.3 g) that was purified by flashchromatography (5) to afford B as an oil (0.85 g; 2.15 mmol). Yield 63%.

C.4-[N,N′-Bis[2-[(9-H-fluoren-9-ylmethoxy)carbonyl]aminoethyl]amino]-4-oxobutanoicacid, D (FIG. 13A)

Succinic anhydride (2 g; 20 mmol) was added in a solution of A (5 g;16.94 mmol) in THF (25 mL) at room temperature. After 28 h the crude wasconcentrated to residue (7 g), washed in ethyl acetate (100 mL) and in 1N HCl (2×50 mL). The organic layer was dried on Na₂SO₄, filtered andevaporated to give crude B as an oily residue (6.53 g). 2 N NaOH (25 mL)was added to suspension of crude B (5 g) in EtOH (35 mL) obtaining acomplete solution after 1 h at room temperature. After 20 h the solventwas evaporated to obtain C as an oil (8.48 g).

A solution of 9-fluorenylmethyl chloroformate (6.54 g, 25.3 mmol) in1,4-dioxane (30 mL), was dropped in the solution of C in 10% aq. Na₂CO₃(30 mL) in 1 h at 0° C. After 20 h at r.t. a gelatinous suspension wasobtained and filtered to give a white solid (3.5 g) and a yellowsolution. The solution was evaporated and the remaining aqueous solutionwas diluted in H₂O (150 mL) and extracted with EtOAc (70 mL). FreshEtOAc (200 mL) was added to aqueous phase, obtaining a suspension whichwas cooled to 0° C. and acidified to pH 2 with conc. HCl. The organiclayer was washed with H₂O (5×200 mL) until neutral pH, then dried togive a glassy solid (6.16 g). The compound was suspended in boilingn-Hexane (60 mL) for 1 h, filtered to give D as a white solid (5.53 g,8.54 mmol). Overall yield 50%.

D.N-[4-[[4-[Bis[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethyl]amino-1,4-dioxobutyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L244 (FIG. 13B)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionemptied and fresh 50% morpholine in DMA (7 mL) was added. The suspensionwas stirred for another 20 min then the solution was emptied and theresin washed with DMA (5×7 mL). 4-[N,N′-Bis[2-[(9-H-fluoren-9-ylmethoxy)carbonyl]aminoethyl]amino]-4-oxo butanoic acid (777.3 mg; 1.2mmol), HOBt (184 mg; 1.2 mmol), DIC (187 μL; 1.2 mmol) and DMA (7 mL)were added to the resin, the mixture shaken for 40 h at roomtemperature, emptied and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for 20 min. The solution was emptied and the resin washedwith DMA (2×7 mL) and with CH₂Cl₂ (5×7 mL) then it was shaken in a flaskwith Reagent B (25 mL) for 4.5 h. The resin was filtered and thesolution was evaporated under reduced pressure to afford an oily crudethat after treatment with Et₂O (20 mL) gave a precipitate. Theprecipitate was collected by centrifugation and washed with Et₂O (5×20mL) to give F as a white solid (140 mg). DOTA tri-t-butyl ester (112 mg;0.178 mmol) HATU (70 mg; 0.178 mmol) and DIEA (60 μL; 0.356 mmol) wereadded to a solution of F (50 mg; 0.0445 mmol) in DMA (3 mL) and CH₂Cl₂(2 mL) and stirred for 24 h at room temperature. The crude wasevaporated to reduced volume (1 mL) and shaken with Reagent B (25 mL)for 4.5 h. After evaporation of the solvent, the residue was treatedwith Et₂O (20 mL) to give a precipitate. The precipitate was collectedby centrifugation and washed with Et₂O (5×20 mL) to afford a beige solid(132 mg) that was analyzed by HPLC. An amount of crude (100 mg) waspurified by preparative HPLC. The fractions containing the product werelyophilized to give L244 (FIG. 13C) (3.5 mg; 1.84×10⁻³ mmol) as a whitesolid. Yield 0.8%.

General Experimentals for Examples XIV-Example XLII L201-L228

A. Manual Couplings

6.0 equivalents of the appropriately protected amino acid was treatedwith 6.0 equivalents each of HOBt and DIC and activated outside thereaction vessel. This activated carboxylic acid in NMP was thentransferred to the resin containing the amine and the reaction wascarried out for 4-6 h and then the resin was drained and washed.

B. Special Coupling of Fmoc-Gly-OH to 4-Aminobenzoic Acid andAminobiphenylcarboxylic Acid Amides:

Fmoc-Gly-OH (10.0 equiv.) was treated with HATU (10.0 equiv.) and DIEA(20.0 equiv.) in NMP (10 mL of NMP was used for one gram of the aminoacid by weight) and the solution was stirred for 10-15 min at RT beforetransferring to the vessel containing the amine loaded resin. The volumeof the solution was made to 15.0 ml for every gram of the resin. Thecoupling was continued for 20 h at RT and the resin was drained of allthe reactants. This procedure was repeated one more time and then washedwith NMP before moving on to the next step.

C. Preparation of D03A Monoamide:

8.0 equivalents of DOTA mono acid was dissolved in NMP and treated with8.0 equivalents of HBTU and 16.0 equivalents of DIEA. This solution wasstirred for 15 min at RT and then transferred to the amine on the resinand the coupling was continued for 24 h at RT. The resin was thendrained, washed and then the peptide was cleaved and purified.

D. Cleavage of the Crude Peptides from the Resin and Purification:

The resin was suspended in Reagent B (15.0 ml/g) and shaken for 4 h atRT. The resin was then drained and washed with 2×5 mL of Reagent B againand combined with the previous filtrate. The filtrate was thenconcentrated under reduced pressure to a paste/liquid at RT andtriturated with 25.0 mL of anhydrous ether (for every gram of the resinused). The suspension was then centrifuged and the ether layer wasdecanted. This procedure was repeated two more times and the colorlessprecipitate after ether wash was purified by preparative HPLC.

Example XIV FIG. 21 Synthesis of L201

0.5 g of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-M-Resin (0.4 mmol/g, 0.5 g,0.2 mmol) (Resin A) was used. The rest of the amino acid units wereadded as described in the general procedure to prepare (1R)-1-(Bis{2-[bis(carboxymethyl)amino]ethyl}amino)propane-3-carboxylicacid-1-carboxyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L201), Yield: 17.0 mg (5.4%)

Example XV FIGS. 22A and 22B Synthesis of L202 A.4-Fmoc-hydrazinobenzoic acid (FIG. 22A)

A suspension of 4-hydrazinobenzoic acid (5.0 g, 32.9 mmol) in water (100ml) was treated with cesium carbonate (21.5 g, 66.0 mmol). Fmoc-Cl (9.1g, 35.0 mmol) in THF (25 mL) was added dropwise to the above solutionwith stirring over a period of 1 h. The solution was stirred for 4 hmore after the addition and the reaction mixture was concentrated toabout 75 mL and extracted with ether (2×100 mL). The ether layer wasdiscarded and the aqueous layer was acidified with 2N HCl. The separatedsolid was filtered, washed with water (5×100 mL) and then recrystallizedfrom acetonitrile to yield the product (compound B) as a colorlesssolid. Yield: 11.0 g (89%). ¹H NMR (DMSO-d₆): δ 4.5 (m, 1H, Ar—CH ₂—CH),4.45 (m, 2H, Ar—CH ₂), 6.6 (bs, 1H, Ar—H), 7.4-7.9 (m, 9, Ar—H and Ar—CH₂), 8.3 (s, 2H, Ar—H), 9.6 (s, 2H, Ar—H). M. S.—m/z 373.2 [M−H]

0.5 g of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-M-Resin (0.4 mmol/g, 0.5 g,0.2 mmol) (Resin A) was used. The amino acid units were added asdescribed in the general procedure, including Compound B to prepareN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-4-hydrazinobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L202) (FIG. 22B), Yield: 25.0 mg (8.3%)

Example XVI FIGS. 23A and 23B Synthesis of L203 A. Preparation of4-Boc-aminobenzyl benzoate Compound B (FIG. 23A)

A suspension of 4-boc-aminobenzoic acid (0.95 g, 4.0 mmol) in dryacetonitrile (10.0 mL) was treated with powdered cesium carbonate (1.3g, 4.0 mmol) and stirred vigorously under nitrogen. Benzyl bromide (0.75g, 4.4 mmol) was added and the reaction mixture was refluxed for 20 hunder nitrogen. The reaction mixture was then poured into ice cold water(200 mL) and the solid separated was filtered and washed with water(5×50 mL). The crude material was then recrystallized from aqueousmethanol to yield the product as a colorless solid (Compound B). Yield:0.8 g (61%). ¹H NMR (CDCl₃): δ 1.5 (s, 9H, Tertiary methyls), 5.4 (s,2H, Ar—CH ₂), 7.4 (m, 7H, Ar—H) and 8.0 (m, 2H, Ar—H). M. S.—m/z 326.1[M+H].

B. 4-Aminobenzyl benzoate Compound C (FIG. 23A)

4-Boc-aminobenzyl benzoate (0.8 g, 2.5 mmol) was dissolved in DCM (20mL) containing TFA (25% by volume) and stirred for 2 h at RT. Thereaction mixture was poured into 100.0 g of crushed ice and neutralizedwith saturated sodium bicarbonate solution until the pH reached about8.5. The organic layer was separated and the aqueous layer was extractedwith DCM (3×20 mL) and all the organic layers were combined. The DCMlayer was then washed with 1×50 mL of saturated sodium bicarbonate,water (2×50 mL) and dried (sodium sulfate). Removal of the solventyielded a colorless solid (Compound C) that was taken to the next stepwithout further purification. Yield: 0.51 g (91%). ¹H NMR (CDCl₃): δ 5.3(s, 2H, Ar—CH ₂), 6.6 (d, 2H, Ar—H, j=1.0 Hz), 7.4 (m, 5H, Ar—H, J=1.0Hz) and 7.9 (d, 2H, Ar—H, J=1.0 Hz).

C. 4-(2-Chloroacetyl)aminobenzyl benzoate Compound D (FIG. 23A)

The amine (0.51 g, 2.2 mmol) was dissolved in dry dimethylacetamide (5.0mL) and cooled in ice. Chloroacetyl chloride (0.28 g, 2.5 mmol) wasadded dropwise via a syringe and the solution was allowed to come to RTand stirred for 2 h. An additional, 2.5 mmol of chloroacetyl chloridewas added and stirring was continued for 2 h more. The reaction mixturewas then poured into ice cold water (100 mL). The precipitated solid wasfiltered and washed with water and then recrystallized from hexane/etherto yield a colorless solid (Compound D). Yield: 0.38 g (56%). ¹H NMR(CDCl₃): δ 4.25 (s, 2H, CH ₂—Cl), 5.4 (s, 2H, Ar—H), 7.4 (m, 5H, Ar—H),7.6 (d, 2H, Ar—H), 8.2 (d, 2H, Ar—H) and 8.4 (s, 1H, —CONH).

tert-Butyl2-{1,4,7,10-tetraaza-7,10-bis{[(tert-butyl)oxycarbonyl]methyl}-4-[(N-{4-[benzyloxycarbonyl]phenyl}carbamoyl]cyclododecyl}acetate,Compound E (FIG. 23A)

DO3A-tri-t-butyl ester.HCl (5.24 g, 9.5 mmol) was suspended in 30.0 mLof dry acetonitrile and anhydrous potassium carbonate (2.76 g, 20 mmol)was added and stirred for 30 min. The chloroacetamide D (2.8 g, 9.2mmol) in dry acetonitrile (20.0 mL) was then added dropwise to the abovemixture for 10 min. The reaction mixture was then stirred overnight. Thesolution was filtered and then concentrated under reduced pressure to apaste. The paste was dissolved in about 200.0 mL of water and extractedwith 5×50 mL of ethyl acetate. The combined organic layer was washedwith water (2×100 mL) and dried (sodium sulfate). The solution wasfiltered and evaporated under reduced pressure to a paste and the pastewas chromatographed over flash silica gel (600.0 g). Elution with 5%methanol in DCM eluted the product. All the fractions that werehomogeneous on TLC were pooled and evaporated to yield a colorless gum.The gum was recrystallized from isopropylether and DCM to prepareCompound E. Yield: 4.1 g (55%). ¹H NMR (CDCl₃): δ 1.5 (s, 27H, methyls),2.0-3.75 (m, 24H, NCH ₂s), 5.25 (d, 2H, Ar—CH ₂), 7.3 (m, 5H, Ar—H), 7.8(d, 2H, Ar—H) and 7.95 (d, 2H, Ar—H). M. S.—m/z 804.3 [M+H].

D. Reduction of the Above Acid E to Prepare Compound F, (FIG. 23 a)

The benzyl ester E from above (1.0 g, 1.24 mmol) was dissolved inmethanol-water mixture (10.0 mL, 95:5) and palladium on carbon was added(10%, 0.2 g). The solution was then hydrogenated using a Parr apparatusat 50.0 psi for 8 h. The solution was filtered off the catalyst and thenconcentrated under reduced pressure to yield a colorless fluffy solid F.It was not purified further and was taken to the next step immediately.MS: m/z 714.3 [M+Na].

E. Preparation of L203 (FIG. 23B)

The above acid F was coupled to the amine on the resin[H-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-Resin] Resin A and F from above usingstandard coupling procedures described above. 0.5 g (0.2 mmol) of theresin yielded 31.5 mg of the final purified peptide (10.9%)N-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L203) (FIG. 23B).

Example XVII FIG. 24 Synthesis of L204

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol) (Resin A)was used. Fmoc-Gly-OH was loaded first followed by F from the aboveprocedure (FIG. 23A) employing standard coupling conditions. Yield: 24.5mg (8.16%) ofN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L204) (FIG. 24).

Example XVIII FIG. 25 Synthesis of L205

Fmoc-6-aminonicotinic acid¹ was prepared as described in the literature(“Synthesis of diacylhydrazine compounds for therapeutic use”.Hoelzemann, G.; Goodman, S. (Merck Patent G.m.b.H., Germany). Ger.Offen.2000, 16 pp. CODEN: GWXXBX DE 19831710 A1 20000120) and coupled withpreloaded Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol)Resin A, followed by the other amino groups as above to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L205) Yield: 1.28 mg (0.4%).

Example XIX FIGS. 26A and 26B Synthesis of L206 A.4′-Fmoc-amino-3′-methylbiphenyl-4-carboxylic acid B

The amino acid (0.41 g, 1.8 mmol) was dissolved in a solution of cesiumcarbonate (0.98 g, 3.0 mmol) in 10.0 mL of water. See “Rational Designof Diflunisal Analogues with Reduced Affinity for Human Serum Albumin”Mao, H. et al J. Am. Chem. Soc., 2001, 123(43), 10429-10435. Thissolution was cooled in an ice bath and a solution of Fmoc-Cl (0.52 g,2.0 mmol) in THF (10.0 mL) was added dropwise with vigorous stirring.After the addition, the reaction mixture was stirred at RT for 20 h. Thesolution was then acidified with 2N HCl. The precipitated solid wasfiltered and washed with water (3×20 mL) and air dried. The crude solidwas then recrystallized from acetonitrile to yield a colorless fluffysolid B (FIG. 26A). Yield: 0.66 g (75%). ¹H NMR (DMSO-d₆): δ 2.2 (s,Ar—Me), 4.25 (t, 1H, Ar—CH, j=5 Hz), 4.5 (d, 2H, O—CH2, j=5.0 Hz), 7.1(bs, 1H, CONH), 7.4-8.0 (m, 8H, Ar—H) and 9.75 (bs, 1H, —COOH). M. S.:m/z 472.0 [M−H].

The acid B from above was coupled toFmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) resin Awith the standard coupling conditions. Additional groups were added asabove to prepareN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-[4′-Amino-2′-methylbiphenyl-4-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L206). Yield: 30.5 mg (24%).

Example XX FIGS. 27A-B Synthesis of L207

3′-Fmoc-amino-biphenyl-3-carboxylic acid was prepared from thecorresponding amine using the procedure described above. See “Synthesisof 3′-methyl-4-′-nitrobiphenylcarboxylic acids by the reaction of3-methyl-4-nitrobenzenenediazonium acetate with methyl benzoate”,Boyland, E. and Gorrod, J., J. Chem. Soc., Abstracts (1962), 2209-11.0.7G of the amine yielded 0.81 g of the Fmoc-derivative (58%) (CompoundB, FIG. 27A). ¹H NMR (DMSO-d₆): δ 4.3 (t, 1H, Ar—CH), 4.5 (d, 2H, O—CH₂), 7.25-8.25 (m, 16H, Ar—H) and 9.9 (s, 1H, —COOH). M. S.—m/z 434 [M−H]

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) resin A wascoupled to the above acid B and additional groups as above (FIG. 27B).29.0 mg ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-[3′-amino-biphenyl-3-carboxyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L207) was prepared (23%).

Example XXI FIG. 28 Synthesis of L208

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A wasdeblocked and coupled to terephthalic acid employing HATU as thecoupling agent. The resulting acid on the resin was activated with DICand NHS and then coupled to ethylenediamine. DOTA-mono acid was finallycoupled to the amine on the resin.N-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-[1,2-d]aminoethyl-terephthalyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L208) was prepared for a yield of 17.5 mg (14%)

Example XXII FIGS. 29A-B Synthesis of L209 A. Boc-Glu(G-OBn)-G-OBn

Boc-Glutamic acid (5.0 g, 20.2 mol) was dissolved in THF (50.0 mL) andcooled to 0° C. in an ice bath. HATU (15.61 g, 41.0 mmol) was addedfollowed by DIEA (6.5 g, 50.0 mmol). The reaction mixture was stirred at0° C. for 30 min. Benzyl ester of glycine [8.45 g, 50 mmol, generatedfrom neutralizing benzyl glycine hydrochloride with sodium carbonate andby extraction with DCM and solvent removal] was added in THF (25.0 mL).The reaction mixture was allowed to come to RT and stirred for 20 h atRT. All the volatiles were removed under reduced pressure. The residuewas treated with saturated sodium carbonate solution (100 mL) andextracted with ethyl acetate (3×100 mL). The organic layers werecombined and washed with 1N HCl (2×100 mL) and water (2×100 mL) anddried (sodium sulfate). The solution was filtered and solvent wasremoved under reduced pressure to yield a paste that was chromatographedover flash silica gel (500.0 g). Elution with 2% methanol in DCM yieldedthe product as a colorless paste (Compound B, FIG. 29A). Yield: 8.5 g(74.5%). ¹H NMR (CDCl₃): δ 1.4 (s, 9H, —CH ₃s), 2.0-2.5 (m, 4H, —CH—CH ₂and CO—CH), 4.2 (m, 5H, N—CH ₂—CO), 5.15 (s, 4H, Ar—CH ₂), 5.45 (bs, 1H,Boc-NH), 7.3 (m, 10H, Ar—H) and 7.6 (2bs, 2H, CONH). M. S.—m/z 564.1[M+H]. Analytical HPLC retention time—8.29 min (>97% pure, 20-65% B over15 min).

B. H-Glu(G-OBn)-G-OBn

The fully protected glutamic acid derivative (1.7 g, 3.2 mmol) B fromabove was dissolved in DCM/TFA (4:1, 20 mL) and stirred until thestarting material disappeared on TLC (2 h). The reaction mixture waspoured into ice cold saturated sodium bicarbonate solution (200 mL) andthe organic layer was separated and the aqueous layer was extracted with2×50 mL of DCM and combined with the organic layer. The DCM layer waswashed with saturated sodium bicarbonate (2×100 mL), water (2×100 mL)and dried (sodium sulfate). The solution was filtered and evaporatedunder reduced pressure and the residue was dried under vacuum to yield aglass (Compound C, FIG. 29A) that was taken to the next step withoutfurther purification. Yield: 0.72 g (95%). M. S.—m/z 442.2 [M+H].

C. (DOTA-tri-t-butyl)-Glu-(G-OBn)-G-OBn

The amine C from above (1.33 g, 3 mmol) in anhydrous DCM (10.0 mL) wasadded to an activated solution of DOTA-tri-t-butyl ester [2.27 g, 3.6mmol was treated with HBTU, 1.36 g, 3.6 mmol and DIEA 1.04 g, 8 mmol andstirred for 30 min at RT in 25 mL of dry DCM] and stirred at RT for 20h]. The reaction mixture was diluted with 200 mL of DCM and washed withsaturated sodium carbonate (2×150 mL) and dried (sodium sulfate). Thesolution was filtered and solvent was removed under reduced pressure toyield a brown paste. The crude product was chromatographed over flashsilica gel (500.0 g). Elution with 2% methanol in DCM furnished theproduct as a colorless gum (Compound D, FIG. 29A). Yield: 1.7 g (56.8%).¹H NMR (CDCl₃): δ 1.3 and 1.4 (2s, 9H, three methyls each from the freebase and the sodium adduct of DOTA), 2.0-3.5 (m, 20H, N—CH ₂s and —CH—CH₂ and CO—CH ₂), 3.75-4.5 (m, 13H, N—CH ₂—CO), 5.2 (m, 4H, Ar—CH ₂) and7.25 (m, 10H, Ar—H). M. S. m/z—1018.3 [M+Na] and 996.5 [M+H] and 546.3[M+Na+H]/2. HPLC—Retention Time: 11.24 min (>90%, 20-80% B over 30 min).

D. (DOTA-tri-t-butyl)-Glu-(G-OH)-G-OH

The bis benzyl ester (0.2 g, 0.2 mmol) D from above was dissolved inmethanol-water (20 mL, 9:1) and hydrogenated at 50 psi in the presenceof 10% Pd/C catalyst (0.4 g, 50% by wt. water). After the startingmaterial disappeared on HPLC and TLC (4 h), the solution was filteredoff the catalyst and the solvent was removed under reduced pressure andthe residue was dried under high vacuum for about 20 h (<0.1 mm) toyield the product as a colorless foam (Compound E, FIG. 29A). Yield:0.12 g (73.5%). ¹H NMR (DMSO-d₆): δ 1.3 and 1.4 (2s, 9H corresponding tomethyls of free base and the sodium adduct of DOTA), 1.8-4.7 (m, 33H,NCH ₂s, COCH ₂ and CH—CH ₂ and NH—CH—CO), 8.1, 8.2 and 8.4 (3bs, NHCO).M. S.: m/z—816.3 [M+H] and 838.3 [M+Na]. HPLC Retention Time: 3.52 min(20-80% B over 30 min, >95% pure).

E.H-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol) A wasdeblocked and coupled twice sequentially to 8-amino-3,6-dioxaoctanoicacid to yield the above deprotected peptide (Compound F, FIG. 29B) afterpreparative HPLC purification. Yield: 91.0 mg (37%).

HPLC Retention Time: 8.98 min (>95% purity, 10-40% B in over 10 min). M.S.: m/z—1230.6 [M+H], 615.9 [M+2H]/2.

F. Solution Phase Coupling of the Bis-Acid E and the Amine F from Above:(FIG. 29B)

The bis-acid (13.5 mg, 0.0166 mmol) E was dissolved in 100 μL of dryacetonitrile and treated with NHS (4.0 mg, 0.035 mmol) and DIC (5.05 mg,0.04 mmol) and stirred for 24 h at RT. To the above activated acid, thefree amine F (51.0 mg, 0.41 mmol)[generated from the TFA salt bytreatment with saturated sodium bicarbonate and freeze drying thesolution to yield the amine as a fluffy solid] was added followed by 100μL of NMP and the stirring was continued for 40 h more at RT. Thesolution was diluted with anhydrous ether (10 mL) and the precipitatewas collected by centrifugation and washed with 2×10 mL of anhydrousether again. The crude solid was then purified by preparative HPLC toyield the product as a colorless fluffy solid L209 as in FIG. 29B with ayield of 7.5 mg (14.7%).

Example XXIII FIGS. 30A-B Synthesis of L210 A.H-8-aminooctanoyl-8-aminooctanoyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂

This was also prepared exactly the same way as in the case of Compound F(FIG. 29B), but using 1-aminooctanoic acid and the amine (Compound B,FIG. 30A) was purified by preparative HPLC. Yield: 95.0 mg (38.9%). HPLCRetention Time: 7.49 min (>95% purity; 10-40% B over 10.0 min). M. S.:m/z—1222.7 [M+H], 611.8 [M+2H]/2.

(DOTA-tri-t-butyl)-Glu-(G-OH)-G-OH (0.0163 g, 0.02 mmol) was convertedto its bis-NHS ester as in the case of L209 in 100 μL of acetonitrileand treated with the free base, Compound B (60.0 mg, 0.05 mmol) in 100μL of NMP and the reaction was continued for 40 h and then worked up andpurified as above to prepare L210 (FIG. 30B) for a yield of 11.0 mg(18%).

Example XXIV FIG. 31 Synthesis of L211

Prepared from 0.2 g Of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin(0.08 mmol) using standard protocols.N-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL211 was prepared in a yield of 4.7 mg (3.7%) (FIG. 31).

Example XXV FIG. 32 Synthesis of L212

Prepared from Rink Amide Novagel resin (0.47 mmol/g, 0.2 g, 0.094 mmol)by building the sequence on the resin by standard protocols.N-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutamyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL212 was prepared for a yield of 25.0 mg (17.7%) (FIG. 32).

Example XXVI FIG. 33 Synthesis of L213

Prepared from Fmoc-Met-2-chlorotrityl chloride resin (NovaBioChem, 0.78mmol/g, 0.26 g, 0.2 mmol) and the rest of the sequence were built usingstandard methodology.N-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionineL213 was prepared for a yield of 49.05 mg (16.4%) (FIG. 33).

Example XXVII FIG. 34 Synthesis of L214

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was usedto prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL214 using standard conditions. 8.5 mg of the product (6.4%) wasobtained (FIG. 34).

Example XXVIII FIG. 35 Synthesis of L215

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was usedto prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-arginyl-L-leucyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL215. 9.2 mg (5.5%) was obtained (FIG. 35).

Example XXIX FIG. 36 Synthesis of L216

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was usedto prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-arginyl-L-tyrosinyl-glycyl-L-asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL216. 25.0 mg (14.7%) was obtained (FIG. 36).

Example XXX FIG. 37 Synthesis of L217

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin A (0.2 g, 0.08 mmol) was usedto prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-lysyl-L-tyrosinyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamideL217. 58.0 mg (34.7%) was obtained (FIG. 37).

Example XXXI FIG. 38 Synthesis of L218

Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin A (0.2 g, 0.08 mmol) was used.Fmoc-Lys(ivDde) was employed for the introduction of lysine. After thelinear sequence was completed, the protecting group of the lysine wasremoved using 10% hydrazine in DMF (2×10 mL; 10 min each and thenwashed). The rest of the amino acids were then introduced usingprocedures described in the “general” section to complete the requiredpeptide sequence. L218 in FIG. 38 as obtained in a yield of 40.0 mg(23.2%).

Example XXXII FIG. 39 Synthesis of L219

4-Sulfamylbutyryl AM Novagel resin was used (1.1 mmol/g; 0.5 g; 0.55mmol). The first amino acid was loaded on to this resin at −20° C. for20 h. The rest of the sequence was completed utilizing normal couplingprocedures. After washing, the resin was alkylated with 20.0 eq. ofiodoacetonitrile and 10.0 equivalents of DIEA for 20 h. The resin wasthen drained of the liquids and washed and then cleaved with 2.0 eq. ofpentylamine in 5.0 mL of THF for 20 h. The resin was then washed with2×5.0 mL of THF and all the filtrates were combined. THF was thenevaporated under reduced pressure and the residue was then deblockedwith 10.0 mL of Reagent B and the peptideN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-aminopentyl,L219 was purified as previously described. 28.0 mg (2.8%) was obtained(FIG. 39).

Example XXXIII FIG. 40 Synthesis of L220

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-D-alanyl-L-histidyl-L-leucyl-L-methioninamide,L220. 31.5 mg (41.4%) was obtained (FIG. 40).

Example XXXIV FIG. 41 Synthesis of L221

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-leucinamide,L221. 28.0 mg (34.3%) was obtained (FIG. 41).

Example XXXV FIG. 42 Synthesis of L222

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-tyrosinyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide,L222. 34.0 mg (40.0%) was obtained (FIG. 42).

Example XXXVI FIG. 43 Synthesis of L223

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide,L223. 31.2 mg (37.1%) was obtained (FIG. 43).

Example XXXVII FIG. 44 Synthesis of L224

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-glycyl-L-histidyl-L-phenylalanyl-L-leucinamide,L224. 30.0 mg (42.2%) was obtained (FIG. 44).

Example XXXVIII FIG. 45 Synthesis of L225

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-valinyl-glycyl-L-serinyl-L-phenylalanyl-L-methioninamide,L225. 15.0 mg (20.4%) was obtained (FIG. 45).

Example XXXIX FIG. 46 Synthesis of L226

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-histidyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L226. 40.0 mg (52.9%) was obtained (FIG. 46).

Example XL FIG. 47 Synthesis of L227

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-threonyll-glycyl-L-histidyl-L-phenylalanyl-L-methioninamideL227. 28.0 mg (36.7%) was obtained (FIG. 47).

Example XLI FIG. 48 Synthesis of L228

NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to prepareN-[(3β,5β,12α)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-phenylalanyl-L-methioninamide,L228. 26.0 mg (33.8%) was obtained (FIG. 48).

Example XLII Synthesis of Additional GRP Compounds A. General procedurefor the preparation of 4,4′-Aminomethylbiphenylcarboxylic acid (B2) and3,3′-aminomethylbiphenylcarboxylic acid (B3) 1.Methyl-hydroxymethylbiphenylcarboxylates

Commercially available (Aldrich Chemical Co.) 4-hydroxymethylphenylboricacid or 3-hydroxymethylphenylboric acid (1.0 g, 6.58 mmol) was stirredwith isopropanol (10 mL) and 2M sodium carbonate (16 mL) until thesolution became homogeneous. The solution was degassed by passingnitrogen through the solution and then treated with solidmethyl-3-bromobenzoate, or methyl-4-bromobenzoate (1.35 g, 6.3 mmol)followed by the Pd (0) catalyst {[(C₆H₅)₃P]₄Pd; 0.023 g, 0.003 mmol}.The reaction mixture was kept at reflux under nitrogen until thestarting bromobenzoate was consumed as determined by TLC analysis (2-3h). The reaction mixture was then diluted with 250 mL of water andextracted with ethyl acetate (3×50 mL). The organic layers were combinedand washed with saturated sodium bicarbonate solution (2×50 mL) anddried (Na₂SO₄). The solvent was removed under reduced pressure and theresidue was chromatographed over flash silica gel (100 g). Elution with40% ethyl acetate in hexanes yielded the product either as a solid oroil.

Yield:

B2—0.45 g (31%); m. p.—170-171° C.

B3—0.69 g (62%); oil.

¹H NMR (CDCl₃) δ B2—3.94 (s, 3H, —COOCH₃), 4.73 (s, 2H, —CH₂-Ph), 7.475(d, 2H, J=5 Hz), 7.6 (d, 2H, J=10 Hz), 7.65 (d, 2H, J=5 Hz) and 8.09 (d,2H, J=10 Hz).

M. S.—m/e—243.0 [M+H]

B3—3.94 (s, 3H, —COOCH₃), 4.76 (s, 2H, —CH₂—Ph), 7.50 (m, 4H), 7.62 (s,1H), 7.77 (s, 1H), 8.00 (s, 1H) and 8.27 (s, 1H).

M. S.—m/e—243.2 [M+H]

2. Azidomethylbiphenyl Carboxylates

The above biphenyl alcohols (2.0 mmol) in dry dichloromethane (10 mL)were cooled in ice and treated with diphenylphosphoryl azide (2.2 mol)and DBU (2.0 mmol) and stirred under nitrogen for 24 h. The reactionmixture was diluted with water and extracted with ethyl acetate (2×25mL). The organic layers were combined and washed successfully with 0.5 Mcitric acid solution (2×25 mL), water (2×25 mL) and dried (Na₂SO₄). Thesolution was filtered and evaporated under reduced pressure to yield thecrude product. The 4,4′-isomer was crystallized from hexane/ether andthe 3,3′-isomer was triturated with isopropyl ether to remove all theimpurities; the product was homogeneous as determined on TLC analysisand further purification was not required.

Yield:

Methyl-4-azidomethyl-4-biphenylcaroxylate—0.245 g (46%); m. p.—106-108°C.

Methyl-4-azidomethyl-4-biphenylcaroxylate—0.36 g (59%, oil)

¹H NMR (CDCl₃) δ—4,4′-isomer—3.95 (s, 3H, —COOCH₃), 4.41 (s, 2H,—CH₂N₃), 7.42 (d, 2H, J=5 Hz), 7.66 (m, 4H) and 8.11 (d, 2H, J=5 Hz)

3,3′-Isomer—3.94 (s, 3H, —COOCH₃), 4.41 (s, 2H, —CH₂N₃), 7.26-7.6 (m,5H), 7.76 (d, 1H, J=10 Hz), 8.02 (d, 1H, J=5 Hz) and 8.27 (s, 1H).

3. Hydrolysis of the Methyl Esters of Biphenylcarboxylates

About 4 mmol of the methyl esters were treated with 20 mL of 2M lithiumhydroxide solution and stirred until the solution was homogeneous (20-24h). The aqueous layer was extracted with 2×50 mL of ether and theorganic layer was discarded. The aqueous layer was then acidified with0.5 M citric acid and the precipitated solid was filtered and dried. Noother purification was necessary and the acids were taken to the nextstep.

Yield:

4,4′-isomer—0.87 g of methyl ester yielded 0.754 g of the acid (86.6%);m. p.—205-210° C.

3,3′-isomer—0.48 g of the methyl ester furnished 0.34 g of the acid(63.6%); m. p.—102-105° C.

¹H NMR (DMSO-d₆) δ: 4,4′-isomer—4.52 (s, 2H, —CH₂N₃), 7.50 (d, 2H, J=5Hz), 7.9 (m, 4H), and 8.03 (d, 2H, J=10 Hz)

3,3′-isomer—4.54 (s, 2H, —CH₂N₃), 7.4 (d, 1H, J=10 Hz), 7.5-7.7 (m, 4H),7.92 (ABq, 2H) and 8.19 (s, 1H).

4. Reduction of the Azides to the Amine

This was carried out on the solid phase and the amine was neverisolated. The azidocarboxylic acid was loaded on the resin using thestandard peptide coupling protocols. After washing, the resin containingthe azide was shaken with 20 equivalents of triphenylphosphine inTHF/water (95:5) for 24 h. The solution was drained under a positivepressure of nitrogen and then washed with the standard washingprocedure. The resulting amine was employed in the next coupling.

5.(3β,5β,7α,12α)-3-[{(9H-Flouren-9ylmethoxy)amino]acetyl}amino-7,12-dihydroxycholan-24-oicacid

Tributylamine (3.2 mL); 13.5 mmol) was added dropwise to a solution ofFmoc-glycine (4.0 g, 13.5 mmol) in THF (80 mL) stirred at 0° C.Isobutylchloroformate (1.7 mL; 13.5 mmol) was subsequently added and,after 10 min, a suspension of tributylamine (2.6 mL; 11.2 mmol) and(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid (4.5 g; 11.2mmol) in DMF (80 mL) was added dropwise, over 1 h, into the cooledsolution. The mixture was allowed to warm up to ambient temperature andafter 6 h, the solution was concentrated to 120 mL, then water (180 mL)and 1N HCl (30 mL) were added (final pH 1.5). The precipitated solid wasfiltered, washed with water (2×100 mL), vacuum dried and purified byflash chromatography. Elution with chloroform/methanol (8:2) yielded theproduct as a colorless solid.

Yield: 1.9 g (25%). TLC: R_(f) 0.30 (CHCl₃/MeOH/NH₄OH—6:3:1).

In Vitro and In Vivo Testing of Compounds Example XLIII In Vitro BindingAssay for GRP Receptors in PC3 Cell Lines—FIGS. 14 A-B

To identify potential lead compounds, an in vitro assay that identifiescompounds with high affinity for GRP-R was used. Since the PC3 cellline, derived from human prostate cancer, is known to exhibit highexpression of GRP-R on the cell surface, a radio ligand binding assay ina 96-well plate format was developed and validated to measure thebinding of ¹²⁵I-BBN to GRP-R positive PC3 cells and the ability of thecompounds of the invention to inhibit this binding. This assay was usedto measure the IC₅₀ for RP527 ligand, DO3A-monoamide-Aoc-QWAVGHLM-NH₂(SEQ ID NO: 1) (controls) and compounds of the invention which inhibitthe binding of ¹²⁵I-BBN to GRP-R.(RP527=N,N-dimethylglycine-Ser-Cys(Acm)-Gly-5-aminopentanoic acid-BBN(7-14) [SEQ. ID. NO: 1], which has MS=1442.6 and IC50-0.84). Van deWiele C, Dumont F et al., Technetium-99m RP527, a GRP analogue forvisualization of GRP receptor-expressing malignancies: a feasibilitystudy. Eur. J. Nucl. Med., (2000) 27; 1694-1699.;DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1) is also referred to asDO3A-monoamide-8-amino-octanoic acid-BBN (7-14) [SEQ. ID. NO: 1], andhas MS=1467.0. DO3A monoamide-aminooctanyl-BBN[7-14]

The Radioligand Binding Plate Assay was validated for BBN and BBNanalogues (including commercially available BBN and L1) and also using^(99m)Tc RP527 as the radioligand.

A. Materials and Methods:

1. Cell Culture:

PC3 (human prostate cancer cell line) were obtained from the AmericanType Culture Collection and cultured in RPMI 1640 (ATCC) in tissueculture flasks (Corning). This growth medium was supplemented with 10%heat inactivated FBS (Hyclone, SH30070.03), 10 mM HEPES (GibcoBRL,15630-080), and antibiotic/antimycotic (GibcoBRL, 15240-062) for a finalconcentration of penicillin-streptomycin (100 units/mL), and fungizone(0.25 μg/mL). All cultures were maintained in a humidified atmospherecontaining 5% CO₂/95% air at 37° C., and passaged routinely using 0.05%trypsin/EDTA (GibcoBRL 25300-054) where indicated. Cells for experimentswere plated at a concentration of 2.0×10⁴/well either in 96-wellwhite/clear bottom microtiter plates (Falcon Optilux-I) or 96 wellblack/clear collagen I cellware plates (Beckton Dickinson Biocoat).Plates were used for binding studies on day 1 or 2 post-plating.

2. Binding Buffer:

RPMI 1640 (ATCC) supplemented with 20 mM HEPES, 0.1% BSA (w/v), 0.5 mMPMSF (AEBSF), bacitracin (50 mg/500 ml), pH 7.4. ¹²⁵I-BBN (carrier free,2200 Ci/mmole) was obtained from Perkin-Elmer.

B. Competition Assay with ¹²⁵I-BBN for GRP-R in PC3 Cells:

A 96-well plate assay was used to determine the IC₅₀ of variouscompounds of the invention to inhibit binding of ¹²⁵I-BBN to humanGRP-R. The following general procedure was followed:

All compounds tested were dissolved in binding buffer and appropriatedilutions were also done in binding buffer. PC3 cells (human prostatecancer cell line) for assay were plated at a concentration of2.0×10⁴/well either in 96-well white/clear bottomed microtiter plates(Falcon Optilux-I) or 96 well black/clear collagen I cellware plates(Beckton Dickinson Biocoat). Plates were used for binding studies on day1 or 2 post-plating. The plates were checked for confluency (>90%confluent) prior to assay. For the assay, RP527 orDO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1) ligand, (controls), orcompounds of the invention at concentrations ranging from 1.25×10⁻⁹ M to5×10⁻⁹ M, was co-incubated with ¹²⁵I-BBN (25,000 cpm/well). Thesestudies were conducted with an assay volume of 75 μl per well.Triplicate wells were used for each data point. After the addition ofthe appropriate solutions, plates were incubated for 1 h at 4° C. toprevent internalization of the ligand-receptor complex. Incubation wasended by the addition of 200 μl of ice-cold incubation buffer. Plateswere washed 5 times and blotted dry. Radioactivity was detected usingeither the LKB CompuGamma counter or a microplate scintillation counter.

Competition binding curves for RP527 (control) and L70, a compound ofthe invention can be found in FIGS. 14A-B. These data show that the IC50of the RP527 control is 2.5 nM and that of L70, a compound of thisinvention is 5 nM. The IC50 of the DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQID NO: 1) control was 5 nM. IC50 values for those compounds of theinvention tested can be found in Tables 1-3, supra, and show that theyare comparable to that of the controls and thus would be expected tohave sufficient affinity for the receptor to allow uptake by receptorbearing cells in vivo.

C. Internalization & Efflux Assay:

These studies were conducted in a 96-well plate. After washing to removeserum proteins, PC3 cells were incubated with ¹²⁵I-BBN,¹⁷⁷Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1) or radiolabeledcompounds of this invention for 40 min, at 37° C. Incubations werestopped by the addition of 200 μl of ice-cold binding buffer. Plateswere washed twice with binding buffer. To remove surface-boundradioligand, the cells were incubated with 0.2M acetic acid (in saline),pH 2.8 for 2 min. Plates were centrifuged and the acid wash media werecollected to determine the amount of radioactivity which was notinternalized. The cells were collected to determine the amount ofinternalized ¹²⁵I-BBN, and all samples were analyzed in the gammacounter. Data for the internalization assay was normalized by comparingcounts obtained at the various time points with the counts obtained atthe final time point (T40 min).

For the efflux studies, after loading the PC3 cells with ¹²⁵I-BBN orradiolabeled compounds of the invention for 40 min at 37° C., theunbound material was filtered, and the % of internalization wasdetermined as above. The cells were then resuspended in binding bufferat 37° C. for up to 3 h. At 0.5, 1, 2, or 3 h, the amount remaininginternalized relative to the initial loading level was determined asabove and used to calculate the percent efflux recorded in Table 5.

TABLE 5 Internalisation and efflux of ¹²⁵I-BBN and the Lu-177 complexesof DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1) (control) andcompounds of this invention DO3A- monoamide- Aoc- QWAVGHLM- NH₂ (SEQ IDNO: 1) I-BBN (control) L63 L64 L70 Internalisation 59 89 64 69 70 (40minutes) Efflux (2 h) 35 28 0 20 12These data show that the compounds of this invention are internalizedand retained by the PC3 cells to a similar extent to the controls.

Example XLIV Preparation of Tc-Labeled GRP Compounds

Peptide solutions of compounds of the invention identified in Table 6were prepared at a concentration of 1 mg/mL in 0.1% aqueous TFA. Astannous chloride solution was prepared by dissolving SnCl₂.2H₂O (20mg/mL) in 1 N HCl. Stannous gluconate solutions containing 20 μg ofSnCl₂.2H₂O/100 μL were prepared by adding an aliquot of the SnCl₂solution (10 μL) to a sodium gluconate solution prepared by dissolving13 mg of sodium gluconate in water. A hydroxypropyl gamma cyclodextrin[HP-γ-CD] solution was prepared by dissolving 50 mg of HP-γ-CD in 1 mLof water.

The ^(99m)Tc labeled compounds identified below were prepared by mixing20 μL of solution of the unlabeled compounds (20 μg), 50 μL of HP-γ-CDsolution, 100 μL of Sn-gluconate solution and 20 to 50 μL of ^(99m)Tcpertechnetate (5 to 8 mCi, Syncor). The final volume was around 200 μLand final pH was 4.5-5. The reaction mixture was heated at 100° C. for15 to 20 min. and then analyzed by reversed phase HPLC to determineradiochemical purity (RCP). The desired product peaks were isolated byHPLC, collected into a stabilizing buffer containing 5 mg/mL ascorbicacid, 16 mg/mL HP-γ-CD and 50 mM phosphate buffer, pH 4.5, andconcentrated using a speed vacuum to remove acetonitrile. The HPLCsystem used for analysis and purification was as follows: C18 Vydaccolumn, 4.6×250 mm, aqueous phase: 0.1% TFA in water, organic phase:0.085% TFA in acetonitrile. Flow rate: 1 mL/min. Isocratic elution at20%-25% acetonitrile/0.085% TFA was used, depending on the nature ofindividual peptide.

Labeling results are summarized in Table 6.

TABLE 6 HPLC RCP⁴ (%) retention Initial immediately time RCP³ followingCompound¹ Sequence² (min) (%) purification L2 -RJQWAVGHLM-NH₂ 5.47 89.995.6 L4 -SJQWAVGHLM-NH₂ 5.92 65 97 L8 -JKQWAVGHLM-NH₂ 6.72 86 94 L1K-JQWAVGHLM-NH₂ 5.43 88.2 92.6 L9 -JRQWAVGHLM-NH₂ 7.28 91.7 96.2 L7-aJQWAVGHLM-NH₂ 8.47 88.6 95.9 n.d. = not detected ¹All compounds wereconjugated with an N,N′-dimethylglycyl-Ser-Cys-Gly metal chelator. TheAcm protected form of the ligand was used. Hence, the ligand used toprepare the 99mTc complex of L2 was N,N′-dimethylglycyl-Ser-Cys(Acm)-Gly-RJQWAVGHLM-NH₂ (SEQ ID NO: 1). The Acm group was removedduring chelation to Tc. ²In the Sequence, “J” refers to8-amino-3,6-dioxaoctanoic acid and “a” refers to D-alanine and QWAVGHLMis SEQ ID NO: 1. ³Initial RCP measurement taken immediately afterheating and prior to HPL purification. ⁴RCP determined following HPLCisolation and acetonitrile removal via speed vacuum.

Example XLV Preparation of ¹⁷⁷Lu-L64 for Cell Binding andBiodistribution Studies

This compound was synthesized by incubating 10 μg L64 ligand (10 μL of a1 mg/mL solution in water), 100 μL ammonium acetate buffer (0.2M, pH5.2) and ˜1-2 mCi of ¹⁷⁷LuCl₃ in 0.05N HCl (MURR) at 90° C. for 15 min.Free ¹⁷⁷Lu was scavenged by adding 20 μL of a 1% Na₂EDTA.2H₂O (Aldrich)solution in water. The resulting radiochemical purity (RCP) was ˜95%.The radiolabeled product was separated from unlabeled ligand and otherimpurities by HPLC, using a YMC Basic C8 column [4.6×150 mm], a columntemperature of 30° C. and a flow rate of 1 mL/min, with a gradient of68% A/32% B to 66% A/34% B over 30 min., where A is citrate buffer(0.02M, pH 3.0), and B is 80% CH₃CN/20% CH₃OH. The isolated compound hadan RCP of ˜100% and an HPLC retention time of 23.4 minutes.

Samples for biodistribution and cell binding studies were prepared bycollecting the desired HPLC peak into 1000 μL of citrate buffer (0.05 M,pH 5.3, containing 1% ascorbic acid, and 0.1% HSA). The organic eluentin the collected eluate was removed by centrifugal concentration for 30min. For cell binding studies, the purified sample was diluted withcell-binding media to a concentration of 1.5 μCi/mL within 30 minutes ofthe in vitro study. For biodistribution studies, the sample was dilutedwith citrate buffer (0.05 M, pH 5.3, containing 1% sodium ascorbic acidand 0.1% HSA) to a final concentration of 50 μCi/mL within 30 minutes ofthe in vivo study.

Example XLVI Preparation of ¹⁷⁷Lu-L64 for Radiotherapy Studies

This compound was synthesized by incubating 70 μL64 ligand (70 μL of a 1mg/mL solution in water), 200 μL ammonium acetate buffer (0.2M, pH 5.2)and ˜30-40 mCi of ¹⁷⁷LuCl₃ in 0.05N HCl (MURR) at 85° C. for 10 min.After cooling to room temperature, free ¹⁷⁷Lu was scavenged by adding 20μL of a 2% Na₂EDTA.2H₂O (Aldrich) solution in water. The resultingradiochemical purity (RCP) was ˜95%. The radiolabeled product wasseparated from unlabeled ligand and other impurities by HPLC, using a300VHP Anion Exchange column (7.5×50 mm) (Vydac) that was sequentiallyeluted at a flow rate of 1 mL/min with water, 50% acetonitrile/water andthen 1 g/L aqueous ammonium acetate solution. The desired compound waseluted from the column with 50% CH₃CN and mixed with ˜1 mL of citratebuffer (0.05 M, pH 5.3) containing 5% ascorbic acid, 0.2% HSA, and 0.9%(v:v) benzyl alcohol. The organic part of the isolated fraction wasremoved by spin vacuum for 40 min, and the concentrated solution (˜20-25mCi) was adjusted within 30 minutes of the in vivo study to aconcentration of 7.5 mCi/mL using citrate buffer (0.05 M, pH 5.3)containing 5% ascorbic acid, 0.2% HSA, and 0.9% (v:v) benzyl alcohol.The resulting compound had an RCP of >95%.

Example XLVII Preparation of ¹¹¹In-L64

This compound was synthesized by incubating 10 μL64 ligand (5 μL of a 2mg/mL solution in 0.01 N HCl), 60 μL ethanol, 1.12 mCi of ¹¹¹InCl₃ in0.05N HCl (80 μL) and 155 μL sodium acetate buffer (0.5M, pH 4.5) at 85°C. for 30 min. Free ¹¹¹In was scavenged by adding 20 μL of a 1%Na₂EDTA.2H₂O (Aldrich) solution in water. The resulting radiochemicalpurity (RCP) was 87%. The radiolabeled product was separated fromunlabeled ligand and other impurities by HPLC, using a Vydac C18 column,[4.6×250 mm], a column temperature of 50° C. and a flow rate of 1.5mL/min. with a gradient of 75% A/25% B to 65% A/35% B over 20 min whereA is 0.1% TFA in water, B is 0.085% TFA in acetonitrile. With thissystem, the retention time for ¹¹¹In-L64 is 15.7 min. The isolatedcompound had an RCP of 96.7%.

Example XLVIII Preparation of ¹⁷⁷Lu-DO3A-monoamide-Aoc-OWAVGHLM-NH₂ (SEQID NO: 1) (Control)

A stock solution of peptide was prepared by dissolvingDO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1) ligand (prepared asdescribed in US Application Publication No. 2002/0054855 and WO02/87637, both incorporated by reference) in 0.01 N HCl to aconcentration of 1 mg/mL. ¹⁷⁷Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ IDNO: 1) was prepared by mixing the following reagents in the order shown.

0.2M NH₄OAc, pH 6.8 100 μl Peptide stock, 1 mg/mL, in 0.01N HCl 5 μl¹⁷⁷LuCl₃ (MURR) in 0.05M HCl 1.2 μl (1.4 mCi)

The reaction mixture was incubated at 85° C. for 10 min. After coolingdown to room temperature in a water bath, 20 μl of a 1% EDTA solutionand 20 μl of EtOH were added. The compound was analyzed by HPLC using aC18 column (VYDAC Cat # 218TP54) that was eluted at flow rate of 1mL/min with a gradient of 21 to 25% B over 20 min, where A is 0.1%TFA/H₂O and B is 0.1% TFA/CH₃CN). ¹⁷⁷Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂(SEQ ID NO: 1) was formed in 97.1% yield (RCP) and had a retention timeof ˜16.1 min on this system.

Example XLIX Preparation of ¹⁷⁷Lu-L63

This compound was prepared as described for¹⁷⁷Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1). The compound wasanalyzed by HPLC using a C18 column (VYDAC Cat #218TP54) that was elutedat flow rate of 1 mL/min with a gradient of 30-34% B over 20 min (wheresolvent is A. 0.1% TFA/H₂O and B is 0.1% TFA/CH₃CN). The ¹⁷⁷Lu-L63 thatformed had an RCP of 97.8% and a retention time of ˜14.2 min on thissystem.

Example L Preparation of ¹⁷⁷Lu-L70 for Cell Binding and BiodistributionStudies

This compound was prepared following the procedures described above, butsubstituting L70 (the ligand of Example II). Purification was performedusing a YMC Basic C8 column (4.6×150 mm), a column temperature of 30° C.and a flow rate of 1 mL/min. with a gradient of 80% A/20% B to 75% A/25%B over 40 min., where A is citrate buffer (0.02M, pH 4.5), and B is 80%CH₃CN/20% CH₃OH. The isolated compound had an RCP of 100% and an HPLCretention time of 25.4 min.

Example LI Preparation of ¹⁷⁷Lu-L70 for Radiotherapy Studies

This compound was prepared as described above for L64.

Example LII Preparation of ¹¹¹In-L70 for Cell Binding andBiodistribution Studies

This compound was synthesized by incubating 10 μL70 ligand (10 μL of a 1mg/mL solution in 0.01 N HCl), 180 μL ammonium acetate buffer (0.2M, pH5.3), 1.1 mCi of ¹¹¹InCl₃ in 0.05N HCl (61 μL, Mallinckrodt) and 50 μLof saline at 85° C. for 30 min. Free ¹¹¹In was scavenged by adding 20 μLof a 1% Na₂EDTA.2H₂O (Aldrich) solution in water. The resultingradiochemical purity (RCP) was 86%. The radiolabeled product wasseparated from unlabeled ligand and other impurities by HPLC, using aWaters XTerra C18 cartridge linked to a Vydac strong anion exchangecolumn [7.5×50 mm], a column temperature of 30° C. and a flow rate of 1mL/min. with the gradient listed in the Table below, where A is 0.1 mMNaOH in water, pH 10.0, B is 1 g/L ammonium acetate in water, pH 6.7 andC is acetonitrile. With this system, the retention time for ¹¹¹In-L70 is15 min while the retention time for L70 ligand is 27 to 28 min. Theisolated compound had an RCP of 96%.

Samples for biodistribution and cell binding studies were prepared bycollecting the desired HPLC peak into 500 μL of citrate buffer (0.05 M,pH 5.3, containing 5% ascorbic acid, 1 mg/mL L-methionine and 0.2% HSA).The organic part of the collection was removed by spin vacuum for 30min. For cell binding studies, the purified, concentrated sample wasused within 30 minutes of the in vitro study. For biodistributionstudies, the sample was diluted with citrate buffer (0.05 M, pH 5.3,containing 5% sodium ascorbic acid and 0.2% HSA) to a finalconcentration of 10 μCi/mL within 30 minutes of the in vivo study.

Time, min A B C  0-10 100% 10-11 100-50%   0-50%  11-21  50% 50% 21-2250-0%  0-50% 50% 22-32  50% 50%

Example LIII In Vivo Pharmacokinetic Studies

A. Tracer Dose Biodistribution:

Low dose pharmacokinetic studies (e.g., biodistribution studies) wereperformed using the below-identified compounds of the invention inxenografted, PC3 tumor-bearing nude mice ([Ncr]-Foxn1<nu>). In allstudies, mice were administered 100 μL of ¹⁷⁷Lu-labeled test compound at200 μCi/kg, i.v., with a residence time of 1 and 24 h per group (n=3-4).Tissues were analyzed in an LKB 1282 CompuGamma counter with appropriatestandards.

TABLE 7 Pharmacokinetic comparison at 1 and 24 h in PC3 tumor-bearingnude mice (200 μCi/kg; values as % ID/g) of ¹⁷⁷Lu-177 labeled compoundsof this invention compared to control DO3A- monoamide- Aoc- QWAVGHLM-NH₂ (SEQ ID NO: 1) control L63 L64 L70 Tissue 1 hr 24 hr 1 hr 24 hr 1 hr24 hr 1 hr 24 hr Blood 0.44 0.03 7.54 0.05 1.87 0.02 0.33 0.03 Liver0.38 0.04 12.15 0.20 2.89 0.21 0.77 0.10 Kidneys 7.65 1.03 7.22 0.8410.95 1.45 6.01 2.31 Tumor 3.66 1.52 9.49 2.27 9.83 3.60 6.42 3.50Pancreas 28.60 1.01 54.04 1.62 77.78 6.56 42.34 40.24

Whereas the distribution of radioactivity in the blood, liver andkidneys after injection of L64 and L70 is similar to that of the controlcompound, DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1)), the uptake inthe tumor is much higher at 1 and 24 h for both L64 and L70. L63 alsoshows high tumour uptake although with increased blood and liver valuesat early times. Uptake in the mouse pancreas, a normal organ known tohave GRP receptors is much higher for L64, L70 and L63 than for thecontrol compound DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1).

Example LIV Receptor Subtype Specificity

Currently, four mammalian members of the GRP receptor family are known:the GRP-preferring receptor (GRP-R), neuromedin-B preferring receptor(NMB-R), the bombesin receptor subtype 3 (BB3-R) and the bombesinreceptor subtype 4 (BB4-R). The receptor subtype specificity of¹⁷⁷Lu-L70 was investigated. The results indicate ¹⁷⁷Lu-L70 bindsspecifically to GRP-R and NMB-R, and has little affinity for BB3-R.

The subtype specificity of the Lutetium complex of L70 (here, ¹⁷⁷Lu-L70)(prepared as described supra) was determined by in vitro receptorautoradiography using the procedure described in Reubi et al., “BombesinReceptor Subtypes in Human Cancers: Detection with the UniversalRadioligand ¹²⁵I-[D-Tyr⁶, beta-Ala, Phe¹³, Nle¹⁴]”, Clin. Cancer Res.8:1139-1146 (2002) and tissue samples that had been previously found toexpress only one subtype of GRP receptor, as well as non-neoplastictissues including normal pancreas and colon, as well as chronicpancreatitis (shown below in Table 8a). Human ileal carcinoid tissue wasused as a source for NMB-R, human prostate carcinoma for GRP-R and humanbronchial carcinoid for BB3-R subtype receptors. For comparison,receptor autoradiography was also performed with other bombesinradioligands, such as ¹²⁵I-Tyr⁴-bombesin or a compound known as theUniversal ligand, ¹²⁵I-[DTyr⁶, βAla¹¹, Phe¹³, Nle¹⁴]-BBN(6-14), whichbinds to all three subsets of GRP-R, on adjacent tissue sections. Forfurther discussion, see Fleischmann et al., “Bombesin Receptors inDistinct Tissue Compartments of Human Pancreatic Diseases,” Lab. Invest.80:1807-1817 (2000); Markwalder et al., “Gastin-Releasing PeptideReceptors in the Human Prostate: Relation to Neoplastic Transformation,”Cancer Res. 59:1152-1159 (1999); Gugger et al., “GRP Receptors inNon-Neoplastic and Neoplastic Human Breast,” Am. J. Pathol.155:2067-2076 (1999).

TABLE 8A Detection of bombesin receptor subtypes in various humantissues using different radioligands. Receptor autoradiography Receptorautoradiography using ¹⁷⁷Lu-L70 using standard BN radioligands* Tumor nGRP-R NMB-R BB3 GRP-R NMB-R BB3 Mammary Ca 8 8/8 0/8 0/8 8/8 0/8 0/8Prostate Ca 4 4/4 0/4 0/4 4/4 0/4 0/4 Renal Ca 6 5/6 0/6 0/6 4/6 0/6 0/6Ileal carcinoid 8 0/8 8/8 0/8 0/8 8/8 0/8 Bronchial carcinoid 6 2/6 0/60/6 2/6 0/6 6/6 (weak) (weak) Colon Ca tumor 7 3/7 0/7 0/7 3/7 0/7 0/7(weak) (weak) smooth muscle 7 7/7 0/7 0/7 7/7 0/7 0/7 Pancreas Ca 4 0/40/4 0/4 0/4 0/4 0/4 Chronic 5 5/5 0/5 0/5 5/5 0/5 0/5 pancreatitis(acini) Human pancreas 7 1/7 (weak) 0/7 0/7 0/7 0/7 0/7 (acini) Mousepancreas 4 4/4 0/4 0/4 4/4 0/4 0/4 (acini) *¹²⁵I-[DTyr⁶, βAla¹¹, Phe¹³,Nle¹⁴]-BBN(6-14) and ¹²⁵I-Tyr⁴-BBN.

A seen from Table 8a, all GRP-R-expressing tumors such as prostatic,mammary and renal cell carcinomas, identified as such with establishedradioligands, were also visualized in vitro with ¹⁷⁷Lu-L70. Due to abetter sensitivity, selected tumors with low levels of GRP-R could beidentified with ¹⁷⁷Lu-L70, but not with ¹²⁵I-Tyr⁴-BBN, as shown in Table8a. All NMB-R-expressing tumors identified with established radioligandswere also visualized with ¹⁷⁷Lu-L70. Conversely, none of the BB3 tumorswere detected with ¹⁷⁷Lu-L70. One should not make any conclusion on thenatural incidence of the receptor expression in the various types oftumors listed in Table 8a, as the tested cases were chosen asreceptor-positive in the majority of cases, with only a few selectednegative controls. The normal human pancreas is not labeled with¹⁷⁷Lu-L70, whereas the mouse pancreas is strongly labeled underidentical conditions. Although the normal pancreas is a very rapidlydegradable tissue and one can never completely exclude degradation ofprotein, including receptors, factors suggesting that the human pancreasdata are truly negative include the positive control of the mousepancreas under similar condition and the strongly labeled BB3 found inthe islets of the respective human pancreas, which represent a positivecontrol for the quality of the investigated human pancreas. Furthermore,the detection of GRP-R in pancreatic tissues that are pathologicallyaltered (chronic pancreatitis) indicate that GRP-R, when present, can beidentified under the chosen experimental conditions in this tissue. Infact, ¹⁷⁷Lu-L70 identifies these GRP-R in chronic pancreatitis withgreater sensitivity than ¹²⁵I-Tyr⁴-BBN. While none of the pancreaticcancers had measurable amounts of GRP-R, a few colon carcinomas showed alow density of heterogeneously distributed GRP receptors measured with¹⁷⁷Lu-L70 (Table 8a). It should further be noticed that the smoothmuscles of the colon express GRP-R and were detected in vitro with¹⁷⁷Lu-L70 as well as with the established bombesin ligands.

TABLE 8B Binding affinity of ¹⁷⁵Lu-L70 to the 3 bombesin receptorsubtypes expressed in human cancers. Data are expressed as IC₅₀ in nM(mean ± SEM. n = number of experiments in parentheses). Compound B.NMB-R C. GRP-R BB3 Universal ligand 0.8 ± 0.1 (3) 0.7 ± 0.1 (3) 1.1 ±0.1 (3) ¹⁷⁵Lu-L70 0.9 ± 0.1 (4) 0.8 ± 0.1 (5) >1,000 (3)

As shown in Table 8b, the cold labeled ¹⁷⁵Lu-L70 had a very highaffinity for human GRP and NMB receptors expressed in human tissueswhile it had only low affinity for BB3 receptors. These experiments used¹²⁵I-[DTyr⁶, βAla¹¹, Phe¹³, Nle¹⁴]-BBN(6-14) as radiotracer. Using the¹⁷⁷Lu-labeled L70 as radiotracer, the above mentioned data are herebyconfirmed and extended. All GRP-R-expressing human cancers were verystrongly labeled with ¹⁷⁷Lu-L70. The same was true for allNMB-R-positive tumors. Conversely, tumors with BB3 were not visualized.The sensitivity of ¹⁷⁷Lu-L70 seems better than that of ¹²⁵I-Tyr⁴-BBN orthe ¹²⁵I labeled universal bombesin analog. Therefore, a few tumorsexpressing a low density of GRP-R can be readily identified with¹⁷⁷Lu-L70, while they are not positive with ¹²⁵I-Tyr⁴-BBN. The bindingcharacteristics of ¹⁷⁷Lu-L70 could also be confirmed in non-neoplastictissues. While the mouse pancreas, as control, was shown to express avery high density of GRP-R, the normal human pancreatic acini weredevoid of GRP-R. However, in conditions of chronic pancreatitis GRP-Rcould be identified in acini, as reported previously in Fleischmann etal., “Bombesin Receptors in Distinct Tissue Compartments of HumanPancreatic Diseases”, Lab. Invest. 80:1807-1817 (2000) and tissue, againwith better sensitivity by using ¹⁷⁷Lu-L70 than by using ¹²⁵I-Tyr⁴-BBN.Conversely, the BB₃-expressing islets were not detected with ¹⁷⁷Lu-L70,while they were strongly labeled with the universal ligand, as reportedpreviously in Fleischmann et al., “Bombesin Receptors in Distinct TissueCompartments of Human Pancreatic Diseases”, Lab. Invest. 80:1807-1817(2000). While a minority of colon carcinomas had GRP-R, usually in verylow density and heterogeneously distributed, the normal colonic smoothmuscles expressed a high density of GRP-R.

The results in Tables 8a and 8b indicate that Lu labeled L70 derivativesare expected to bind well to human prostate carcinoma, which primarilyexpresses GRP-R. They also indicate that Lu labeled L70 derivatives arenot expected to bind well to normal human pancreas (which primarilyexpresses the BB3-R receptor), or to cancers which primarily express theBB3-R receptor subtype.

Example LV Radiotherapy Studies

A. Efficacy Studies:

Radiotherapy studies were performed using the PC3 tumor-bearing nudemouse model. In Short Term Efficacy Studies, ¹⁷⁷Lu labeled compounds ofthe invention L64, L70, L63 and the treatment control compoundDO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1) were compared to anuntreated control group. (n=12 for each treatment group for up to 30days, and n=36 for the pooled untreated control group for up to 31days). For all efficacy studies, mice were administered 100 μL of¹⁷⁷Lu-labeled compound of the invention at 30 mCi/kg, i.v, or s.c. understerile conditions. The subjects were housed in a barrier environmentfor the duration of the study. Body weight and tumor size (by calipermeasurement) were collected on each subject 3 times per week for theduration of the study. Criteria for early termination included: death;loss of total body weight (TBW) equal to or greater than 20%; tumor sizeequal to or greater than 2 cm³. Results of the Short Term Efficacy Studyare displayed in FIG. 15A. These results show that animals treated withL70, L64 or L63 have increased survival over the control animals givenno treatment and over those animals given the same dose of theDO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1) control.

Long Term Efficacy Studies were performed with L64 and L70 using thesame dose as before but using more animals per compound (n=46) andfollowing them for up to 120 days. The results of the Long Term EfficacyStudy are displayed in FIG. 15B. Relative to the same controls as before(n=36), both L64 and L70 treatment gave significantly increased survival(p<0.0001) with L70 being better than L64, although not statisticallydifferent from each other (p<0.067).

Example LVI Alternative Preparation of L64 and L70 Using SegmentCoupling

Compounds L64 and L70 can be prepared employing the collection ofintermediates generally represented by A-D (FIG. 19), which themselvesare prepared by standard methods known in the art of solid and solutionphase peptide synthesis (Synthetic Peptides—A User's Guide 1992, Grant,G., Ed. WH. Freeman Co., NY, Chap 3 and Chap 4 pp 77-258; Chan, W. C.and White, P. D. Basic Procedures in Fmoc Solid Phase PeptideSynthesis—A Practical Approach 2002, Chan, W. C. and White, P. D. EdsOxford University Press, New York, Chap. 3 pp 41-76; Barlos, K. andGatos, G. Convergent Peptide Synthesis in Fmoc Solid Phase PeptideSynthesis—A Practical Approach 2002, Chan, W. C. and White, P. D. EdsOxford University Press, New York, Chap. 9 pp 216-228) which areincorporated herein by reference.

These methods include Aloc, Boc, Fmoc or benzyloxycarbonyl-based peptidesynthesis strategies or judiciously chosen combinations of those methodson solid phase or in solution. The intermediates to be employed for agiven step are chosen based on the selection of appropriate protectinggroups for each position in the molecule, which may be selected from thelist of groups shown in FIG. 1. Those of ordinary skill in the art willalso understand that intermediates, compatible with peptide synthesismethodology, comprised of alternative protecting groups can also beemployed and that the listed options for protecting groups shown aboveserves as illustrative and not inclusive, and that such alternatives arewell known in the art.

This is amply illustrated in FIG. 20 which outlines the approach.Substitution of the intermediate C2 in place of C1 shown in thesynthesis of L64, provides L70 when the same synthetic strategies areapplied.

Example LVII FIGS. 49A and 49B Synthesis of L69

Summary: Reaction of (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid A with Fmoc-Cl gave intermediate B. Rink amide resin functionalisedwith the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14])(SEQ ID NO: 1) (A), was sequentially reacted with B,Fmoc-8-amino-3,6-dioxaoctanoic acid and DOTA tri-t-butyl ester. Aftercleavage and deprotection with Reagent B the crude was purified bypreparative HPLC to give L230. Overall yield: 4.2%.

A.(3β,5β,7α,12α)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oicacid, B (FIG. 49A)

A solution of 9-fluorenylmethoxycarbonyl chloride (1.4 g; 5.4 mmol) in1,4-dioxane (18 mL) was added dropwise to a suspension of(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid A (2.0 g; 4.9mmol) (3) in 10% aq. Na₂CO₃ (30 mL) and 1,4-dioxane (18 mL) stirred at0° C. After 6 h stirring at room temperature H2O (100 mL) was added, theaqueous phase washed with Et₂O (2×90 mL) and then 2 M HCl (15 mL) wasadded (final pH: 1.5). The precipitated solid was filtered, washed withH₂O (3×100 mL), vacuum dried and then purified by flash chromatographyto give B as a white solid (2.2 g; 3.5 mmol). Yield 71%.

B.N-[3β,5β,7α,12α)-3-[[[2-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L69 (FIG. 49B)

Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionfiltered and fresh 50% morpholine in DMA (7 mL) was added. Thesuspension was stirred for another 20 min then the solution was filteredand the resin washed with DMA (5×7 mL).(3β,5β,7α,12α)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oicacid B (0.75 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2mmol), N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7mL) were added to the resin, the mixture shaken for 24 h at roomtemperature, emptied and the resin washed with DMA (5×7 mL). The resinwas then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution emptied, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was emptied and theresin washed with DMA (5×7 mL). Fmoc-8-amino-3,6-dioxaoctanoic acid(0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) andDMA (7 mL) were added to the resin. The mixture was shaken for 3 h atroom temperature, emptied and the resin washed with DMA (5×7 mL). Theresin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, thesolution filtered, fresh 50% morpholine in DMA (7 mL) was added and themixture shaken for another 20 min. The solution was filtered and theresin washed with DMA (5×7 mL)1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acidtris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt(0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), N-ethyldiisopropylamine(0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixturewas shaken for 24 h at room temperature, filtered and the resin washedwith DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin wasshaken in a flask with Reagent B (25 mL) (2) for 4.5 h. The resin wasfiltered and the solution was evaporated under reduced pressure toafford an oily crude that after treatment with Et₂O (20 mL) gave aprecipitate. The precipitate was collected by centrifugation and washedwith Et₂O (3×20 mL) to give a solid (248 mg) which was analysed by HPLC.An amount of crude (50 mg) was purified by preparative HPLC. Thefractions containing the product were lyophilised to give L69 (6.5 mg;3.5×10⁻³ mmol) (FIG. 49B) as a white solid. Yield 5.8%.

Example LVIII FIG. 50 Synthesis of L144

Summary: Rink amide resin functionalised with the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14]) (SEQ ID NO: 1) (A) wasreacted with4-[2-hydroxy-3-[4,7,10-tris[2-(1,1-dimethylethoxy)-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]propoxy]benzoicacid. After cleavage and deprotection with Reagent B (2) the crude waspurified by preparative HPLC to give L144. Overall yield: 12%.

A.N-[4-[2-Hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,L144 (FIG. 50)

Resin A (0.4 g; 0.24 mmol) was shaken in a solid phase peptide synthesisvessel with 50% morpholine in DMA (7 mL) for 10 min, the solutionfiltered and fresh 50% morpholine in DMA (7 mL) was added. Thesuspension was stirred for another 20 min then the solution was filteredand the resin washed with DMA (5×7 mL).4-[2-Hydroxy-3-[4,7,10-tris[2-(1,1-dimethylethoxy)-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]propoxy]benzoicacid B (0.5 g; 0.7 mmol), HOBt (0.11 g; 0.7 mmol), DIC (0.11 mL; 0.7mmol)), N-ethyldiisopropylamine (0.24 mL; 1.4 mmol) and DMA (7 mL) wereadded to the resin. The mixture was shaken for 24 h at room temperature,emptied and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) andvacuum dried. The resin was shaken in a flask with Reagent B (25 mL) (2)for 4.5 h. The resin was filtered and the solution was evaporated underreduced pressure to afford an oily crude that after treatment with Et₂O(20 mL) gave a precipitate. The precipitate was collected bycentrifugation and washed with Et₂O (3×20 mL) to give a solid (240 mg)which was analysed by HPLC. An amount of crude (60 mg) was purified bypreparative HPLC. The fractions containing the product were lyophilisedto give L144 (10.5 mg; 7.2×10⁻³ mmol) as a white solid. Yield 12%.

Example LIX Preparation of L300 and ¹⁷⁷Lu-L300

From 0.2 g of Rink amide Novagel resin (0.63 mmol/g, 0.126 mmol), L300(0.033 g, 17%) was obtained after preparative column chromatography. Theretention time was 6.66 minutes. The molecular formula is C₇₂H₉₉N₁₉O₁₈.The calculated molecular weight is 1518.71; 1519.6 observed. Thesequence is DO3A-Gly-Abz4-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH₂ (SEQ IDNO: 10). The structure of L300 is shown in FIG. 51.

L300 (13.9 μg in 13.9 μL of 0.2M pH 4.8 sodium acetate buffer) was mixedwith 150 μL of 0.2M pH 4.8 sodium acetate buffer and 4 μL of ¹⁷⁷LuCl₃(1.136 mCi, Missouri Research Reactor). After 10 min at 100° C., theradiochemical purity (RCP) was 95%. The product was purified on a VydacC18 peptide column (4.6×250 mm, 5 um pore size) eluted at a flow rate of1 mL/min using an aqueous/organic gradient of 0.1% TFA in water (A) and0.085% TFA in acetonitrile (B). The following gradient was used:isocratic 22% B for 30 min, to 60% B in 5 min, hold at 60% B for 5 min.The compound, which eluted at a retention time of 18.8 min., wascollected into 1 mL of an 0.8% human serum albumin solution that wasprepared by adding HSA to a 9:1 mixture of normal saline and AscorbicAcid, Injection. Acetonitrile was removed using a Speed Vacuum (Savant).After purification, the compound had an RCP of 100%.

Example LX Characterization of Linker Specificity in Relation to GRPReceptor Subtypes

Two cell lines, C6, an NMB-R expressing rodent glioblastoma cell lineand PC3, a GRP-R expressing human prostate cancer cell line, were usedin this assay. The affinity of various unlabeled compounds for eachreceptor subtype (NMB-R and GRP-R) was determined indirectly bymeasuring its ability to compete with the binding of ¹²⁵I-NMB or¹²⁵I-BBN to its corresponding receptors in C6 and PC3 cells.

A. Materials and Methods:

1. Cell Culture:

C6 cells were obtained from ATCC(CCL-107) and cultured in F12K media(ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodium bicarbonate,15% horse serum and 2.5% FBS. Cells for the assays were plated at aconcentration of 9.6×10⁴/well in 48 well poly-lysine coated plates(Beckton Dickinson Biocoat). PC3 were obtained from ATCC(CRL-1435) andcultured in RPMI 1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5 g/LSodium bicarbonate, 10 mM HEPES and 10% FBS. Both cultures weremaintained in a humidified atmosphere containing 5% CO₂/95% air at 37°C. PC3 cells for the assays were plated at a concentration of 2.0×10⁴cells/well in 96-well white/clear bottom plates (Falcon Optilux-I).Plates were used for the assays on day 2 of the post-plating.

2. Binding Buffer, and Radio-Ligands:

RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction V, 1.0mMAEBSF (CAS #3087-99-7) and 0.1% Bacitracin (CAS #1405-87-4), pH 7.4.

-   -   Custom made ¹²⁵I-[Tyr⁰]NMB, >2.0 Ci/μmole (Amersham Life        Science) [¹²⁵I-NMB] and commercially available        ¹²⁵I-[Tyr⁴]BBN, >2.0 Ci/μmole (Perkin Elmer Life Science)        [¹²⁵I-BBN] were used as radioligands.

B. In Vitro Assay:

Using a 48-well plate assay system (for C6 study) competitionexperiments were performed using ¹²⁵I-NMB. All of the PC3 studies wereperformed as described in Example XLIII using ¹²⁵I-BBN. Selection ofcompounds for the assay was based on linker subtype. Results are shownin Table 9.

TABLE 9 Number of selected compounds for the assay and their linkersNUMBER OF LINKER TYPE COMPOUNDS Neutral, Basic or combination ofneutral, 8 basic & acidic Linear aliphatic (ω-aminoalkanoic & 4ω-aminoalkoxynoic acid Bile acids (cholic acids) 3 Substituted alanine(cycloalkyl, aromatic 5 and heteroaromatic) Aromatic (aminobenzoic acidand 12 aminoalkyl benzoic acid, biphenyl) Cyclic non-aromatic 5Heterocyclic (aromatic and non-aromatic) 5 Miscellaneous (DOTA-NMB,DOTA-G- 6 Abz4-NMB, DOTA-Abz4-G-NMB, BBN₇₋₁₄, BBN₈₋₁₄, DOTA-BBN₇₋₁₄)

The binding parameters obtained from the studies were analyzed using aone-site competition non-linear regression analysis with GraphPad Prism.The relative affinity of various compounds for NMB-R in C6 cells werecompared with those obtained using commercially available [Tyr⁴]-BBN and[Tyr⁰]-NMB. To distinguish the GRP-R preferring compounds from NMB-Rplus GRP-R preferring compounds, IC₅₀ values obtained for each compoundwas compared with those obtained from [Tyr⁰]-BBN with ¹²⁵]-NMB on C6cells. The cut off point between the two classes of compounds was takenas 10× the IC₅₀ of [Tyr⁴]-BBN. Among the compounds tested, 8 compoundspreferentially bind to GRP-R (as shown in Table 10) while 32 compoundsbind to both GRP-R and NMB-R with similar affinity, and two showpreference for NMB-R.

TABLE 10 The IC₅₀ values obtained from competition experiments using125I-NMB and 125I-BBN IC₅₀ (nM) GRP-R & L # COMPOUND ¹²⁵I-BBN/PC3¹²⁵I-NMB/C6 GRP-R NMB-R na N,N-dimethylglycine-Ser- 10 10.4 — yesCys(Acm)-Gly-SS- QWAVGHLM-NH₂ (SEQ ID NO: 1) na N,N-dimethylglycine-Ser-25 7.9 — yes Cys(Acm)-Gly-G- QWAVGHLM-NH₂ (SEQ ID NO: 1) naN,N-dimethylglycine-Ser- 48 20.2 — yes Cys(Acm)-Gly-GG- QWAVGHLM-NH₂(SEQ ID NO: 1) na N,N-dimethylglycine-Ser- 13 6.4 — yes Cys(Acm)-Gly-KK-QWAVGHLM-NH₂ (SEQ ID NO: 1) na N,N-dimethylglycine-Ser- 2 2.2 — yesCys(Acm)-Gly-SK- QWAVGHLM-NH₂ (SEQ ID NO: 1) na N,N-dimethylglycine-Ser-1.9 2.0 — yes Cys(Acm)-Gly-SR- QWAVGHLM-NH₂ (SEQ ID NO: 1) naN,N-dimethylglycine-Ser- 7.5 24.1 yes — Cys(Acm)-Gly-KS- QWAVGHLM-NH₂(SEQ ID NO: 1) na N,N-dimethylglycine-Ser- 32 60.0 yes —Cys(Acm)-Gly-KE- QWAVGHLM-NH₂ (SEQ ID NO: 1) na DO3A-monoamide-Aoc- 3.43.1 — yes QWAVGHLM-NH2 (SEQ ID NO: 1) na DO3A-monoamide-Apa3- 36 18.9 —yes QWAVGHLM-NH₂ (SEQ ID NO: 1) na DO3A-monoamide-Abu4- 19.8 5.2 — yesQWAVGHLM-NH₂ (SEQ ID NO: 1) L3 N,N-dimethylglycine-Ser- 70 33 — yesCys(Acm)-Gly-DJ- QWAVGHLM-NH₂ (SEQ ID NO: 1) L64 DO3A-monoamide-G-Adca3-8.5 3.3 — yes QWAVGHLM-NH2 (SEQ ID NO: 1) L63 DO3A-monoamide-G-Ah12ca-23 3.8 — yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L67 DO3A-monoamide-G-Akca- 5.52.3 — yes QWAVGHLM-NH₂ (SEQ ID NO: 1) na DO3A-monoamide-Cha-Cha- 22 77yes — QWAVGHLM-NH₂ (SEQ ID NO: 1) na DO3A-monoamide-Na11-Bip- 30 210.9yes — QWAVGHLM-NH₂ (SEQ ID NO: 1) na DO3A-monoamide-Cha-Na11- 8 66.5 yes— QWAVGHLM-NH₂ (SEQ ID NO: 1) na DO3A-monoamide-Na11-Bpa4- 17 89.9 yes —QWAVGHLM-NH₂ (SEQ ID NO: 1) L301 DO3A-monoamide-Amb4- 10 6.8 yesNa11-QWAVGHLM-NH₂ (SEQ ID NO: 1) L147 DO3A-monoamide-G- 4 32 yes —Mo3abz4-QWAVGHLM-NH₂ (SEQ ID NO: 1) L241 DO3A-monoamide-G- 4 0.8 — yesC13abz4QWAVGHLM-NH₂ (SEQ ID NO: 1) L242 DO3A-monoamide-G-M3abz4- 5 2.2 —yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L243 DO3A-monoamide-G- 14 9.9 — yesHo3abz4-QWAVGHLM-NH₂ (SEQ ID NO: 1) L202 DO3A-monoamide-G-Hybz4- 13 2.7— yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L204 DO3A-monoamide-Abz4-G- 50 1.2 —yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L233 DO3A-monoamide-G-Abz3- 4.8 1.6 —yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L235 DO3A-monoamide-G-Nmabz4- 7 1.5 —yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L147 DO3A-monoamide-Mo3amb4- 3.5 1.2 —yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L71 DO3A-monoamide-Amb4- 7.2 0.2 — yesQWAVGHLM-NH₂ (SEQ ID NO: 1) L73 DO3A-monoamide-Aeb4- 5 1.8 — yesQWAVGHLM-NH₂ (SEQ ID NO: 1) L208 DO3A-monoamide-Dae-Tpa- 8 0.9 — yesQWAVGHLM-NH₂ (SEQ ID NO: 1) L206 DO3A-monoamide-G- 5 1.3 — yesA4m2biphc4-QWAVGHLM- NH₂ (SEQ ID NO: 1) L207 DO3A-monoamide-G- 3 15.1 —yes A3biphc3-QWAVGHLM-NH₂ (SEQ ID NO: 1) L72 DO3A-monoamide-Amc4- 8.22.6 — yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L107 DO3A-monoamide-Amc4- 5 0.3 —yes Amc4-QWAVGHLM-NH₂ (SEQ ID NO: 1) L89 DO3A-monoamide-Aepa4- 23 114yes — QWAVGHLM-NH₂ (SEQ ID NO: 1) L28 N,N-dimethylglycine-Ser- 25 13 —yes Cys(Acm)-Gly-Aepa4-S- QWAVGHLM-NH₂ (SEQ ID NO: 1) L74DO3A-monoamide-G-Inp- 6.5 3.4 — yes QWAVGHLM-NH₂ (SEQ ID NO: 1) L36N,N-dimethylglycine-Ser- 7 12.1 — yes Cys(Acm)-Gly-Pia1-J- QWAVGHLM-NH₂(SEQ ID NO: 1) L82 DO3A-monoamide-Ckbp- 8 1.7 — yes QWAVGHLM-NH₂ (SEQ IDNO: 1) na DO3A-monoamide-Aoc- 11 1.4 — yes QWAVGHL-Nle-NH₂ (SEQ IDNO: 1) L70 DO3A-monoamide-G-Abz4- 4.5 1.5 — yes QWAVGHLM-NH₂ (SEQ IDNO: 1) na DO3A-monoamide- 366 >250 No selective preference QWAVGHLM-NH₂(SEQ ID NO: 1) na QWAVGHLM-NH₂ 369 754 No selective preference (SEQ IDNO: 1) na WAVGHLM-NH₂ >800 >800 No selective preference (SEQ ID NO: 1)L204 DO3A-monoamide-Abz4-G- >50 1.2 preference to NMB-R QWAVGHLM-NH₂(SEQ ID NO: 1) na GNLWATGHFM-NH₂ >500 0.7 preference to NMB-R (SEQ IDNO: 20) L227 DO3A-monoamide-G-Abz4- 28 0.8 — Yes LWATGHFM-NH₂ (SEQ IDNO: 17) In the above Table “na” indicates “not applicable” (e.g. thecompound does not contain a linker of the invention and thus was notassigned an L#).

Based on the above, several results were observed. The receptor bindingregion alone (BBN₇₋₁₄ or BBN₈₋₁₄) did not show any preference to GRP-Ror NMB-R. The addition of a chelator alone to the receptor bindingregion did not contribute to the affinity of the peptide to GRP-R orNMB-R (DO3A-monoamide-QWAVGHLM-NH2 (SEQ ID NO: 1)). Coupling thechelator to the peptide through a linker did contribute to the affinityof the peptide towards the receptor. However, depending on the type oflinker this affinity varied from being dual (preference for both NMB-Rand GRP-R) to GRP-R (preferring GRP-R).

The ω-Aminoalkanoic acids tested (8-Aminooctanoic acid in¹⁷⁵Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH2 (SEQ ID NO: 1) andDO3A-monoamide-Aoc-QWAVGHL-Nle-NH₂ (SEQ ID NO: 1), 3-aminopropionic acidin DO3A-monoamide-Apa3-QWAVGHLM-NH2 (SEQ ID NO: 1) and 4-aminobutanoicacid in DO3A-monoamide-Abu4-QWAVGHLM-NH2 (SEQ ID NO: 1)) as linkers,conferred the peptide with dual affinity for both GRP-R and NMB-R.Replacement of ‘Met’ in ¹⁷⁵Lu-DOTA-Aoc-QWAVGHLM-NH2 (SEQ ID NO: 1) by‘Nle’ did not change this dual affinity of the peptide.

Cholic acid containing linkers (3-aminocholic acid in L64,3-amino-12-hydroxycholanic in L63 and 3-amino-12-ketocholanic in L67conferred the peptides with dual affinity for both GRP-R and NMB-R.Cycloalkyl and aromatic substituted alanine containing linkers(3-cyclohexylalanine in DO3A-monoamide-Cha-Cha-QWAVGHLM-NH2 (SEQ ID NO:1), 1-Naphthylalanine in DO3A-monoamide-Cha-Na11-QWAVGHLM-NH2 (SEQ IDNO: 1), 4-Benzoylphenylalanine in DO3A-monoamide-Na11-Bpa4-QWAVGHLM-NH2(SEQ ID NO: 1) and Biphenylalanine inDO3A-monoamide-Na11-Bip-QWAVGHLM-NH2 (SEQ ID NO: 1)) imparted thepeptides with selective affinity towards GRP-R. A linker containing only4-(2-Aminoethylpiperazine)-1 also contributed to the peptides with GRP-Rselectivity (L89).

Introduction of G-4-amino benzoic acid linker to NMB sequence conferredthe compound with an affinity to GRP-R in addition to its inherent NMB-Raffinity (L227 vs GNLWATGHFM-NH₂) (SEQ ID NO: 20). Shifting the positionof Gly around the linker altered the affinity of L70 from its dualaffinity to a selective affinity to NMB-R (L204). 3-methoxy substitutionin 4-aminobenzoic acid in L70 (as in L240) changed the dual affinity toa selective affinity to GRP-R.

It is apparent from the preceding data that the linker has a significanteffect on the receptor subtype specificity. Three groups of compoundscan be identified:

-   -   Those that are active at the GRP-R

These compounds provide information specific to this receptor in vitroand in vivo, which can be used for diagnostic purposes. When thesecompounds are radiolabeled with a therapeutic radionuclide, therapy canbe performed on tissues containing only this receptor, sparing thosethat contain the NMB-R

-   -   Those that are active at the NMB-R

These compounds provide information specific to this receptor in vitroand in vivo, which can be used for diagnostic purposes. Whenradiolabeled with a therapeutic radionuclide, therapy can be performedon tissues containing only this receptor, sparing those that contain theGRP-R

-   -   Those that are active at both the GRP-R and the NMB-R

These compounds provide information on the combined presence of thesetwo receptor subtypes in vitro and in vivo, that can be used fordiagnostic purposes. Targeting both receptors may increase thesensitivity of the examination at the expense of specificity. When thesecompounds are radiolabeled with a therapeutic radionuclide, therapy canbe performed on tissues containing both receptors, which may increasethe dose delivered to the desired tissues.

Example LXI Competition Studies of Modified Bombesin (BBN) Analogs with¹²⁵I-BBN for GRP-R in Human Prostate Cancer (PC3) Cells

To determine the effect of replacing certain amino acids in the BBN 7-14analogs, peptides modified in the targeting portion were made andassayed for competitive binding to GRP-R in human prostate cancer (PC3)cells. All these peptides have a common linker conjugated to a metalchelating moiety (DOTA-Gly-Abz-4-). The binding data (IC₅₀ nM) are givenbelow in Table 13.

A. Materials and Methods:

1. Cell Culture:

PC3 cell lines were obtained from ATCC(CRL-1435) and cultured in RPMI1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodiumbicarbonate, 10 mM HEPES and 10% FBS. Cultures were maintained in ahumidified atmosphere containing 5% CO₂/95% air at 37° C. PC3 cells forthe assays were plated at a concentration of 2.0×10⁴ cells/well in a96-well white/clear bottom plates (Falcon Optilux-I). Plates were usedfor the assays on day 2 of the post-plating.

2. Binding Buffer:

RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction V, 1.0 mMAEBSF (CAS #3087-99-7) and 0.1% Bacitracin (CAS #1405-87-4), pH 7.4.

3. ¹²⁵I-Tyr⁴-Bombesin [¹²⁵I-BBN]

¹²⁵I-BBN (Cat # NEX258) was obtained from PerkinElmer Life Sciences.

C. In Vitro Assay:

Competition assay with ¹²⁵I-BBN for GRP-R in PC3 cells:

All compounds tested were dissolved in binding buffer and appropriatedilutions were also done in binding buffer. PC3 cells for assay wereseeded at a concentration of 2.0×10⁴/well either in 96-well black/clearcollagen I cellware plates (Beckton Dickinson Biocoat). Plates were usedfor binding studies on day 2 post-plating. The plates were checked forconfluency (>90% confluent) prior to assay. For competition assay,N,N-dimethylglycyl-Ser-Cys(Acm)-Gly-Ava5-QWAVGHLM-NH₂ (SEQ ID NO: 1)(control) or other competitors at concentrations ranging from 1.25×10⁻⁹Mto 500×10⁻⁹ M, was co-incubated with ¹²⁵I-BBN (25,000 cpm/well). Thestudies were conducted with an assay volume of 75 μl per well.Triplicate wells were used for each data point. After the addition ofthe appropriate solutions, plates were incubated for 1 hour at 4° C.Incubation was ended by the addition of 200 uL of ice-cold incubationbuffer. Plates were washed 5 times and blotted dry. Radioactivity wasdetected using either a LKB CompuGamma counter or a microplatescintillation counter. The bound radioactivity of ¹²⁵I-BBN was plottedagainst the inhibition concentrations of the competitors, and theconcentration at which ¹²⁵I-BBN binding was inhibited by 50% (IC₅₀) wasobtained from the binding curve.

TABLE 13 Competition studies with ¹²⁵I-BBN for GRP-R in PC3 cells L #PEPTIDES IC₅₀ [nM] Ref na N,N-dimethylglycyl- 2.5 Ser-Cys(Acm)-Gly-Ava5-QWAVGHLM-NH₂ (SEQ ID NO: 1) 1 L70 DO3A-monoamide-G- 4.5Abz4-QWAVGHLM-NH₂ (SEQ ID NO: 1) 2 L214 DO3A-monoamide-G- 18Abz4-fQWAVGHLM-NH₂ (SEQ ID NO: 1) 3 L215 DO3A-monoamide-G- 6Abz4-QRLGNQWAVGHLM-NH₂ (SEQ ID NO: 3) 4 L216 DO3A-monoamide-G- 4.5Abz4-QRYGNQWAVGHLM-NH₂ (SEQ ID NO: 4) 5 L217 DO3A-monoamide-G- 10Abz4-QKYGNQWAVGHLM-NH₂ (SEQ ID NO: 5) 6 L218 >EQ-[K(DO3A-monoamide-G- 53Abz4)-LGNQWAVGHLM-NH₂ (SEQ ID NO: 18) 7 L219 DO3A-monoamide-G-Abz4- 75fQWAVGHLM-NH—C₅H₁₂ (SEQ ID NO: 1) 8 L220 DO3A-monoamide-G- 13Abz4-QWAVaHLM-NH₂ (SEQ ID NO: 15) 9 L221 DO3A-monoamide-G- 340Abz4-fQWAVGHLL-NH₂ (SEQ ID NO: 8) 10 L222 DO3A-monoamide-G-Abz4- 46yQWAV-Ala2-HF-Nle-NH₂ (SEQ ID NO: 10) 11 L223 DO3A-monoamide-G-Abz4- 52FQWAV-Ala2-HF-Nle-NH₂ (SEQ ID NO: 21) 12 L224 DO3A-monoamide-G- >500Abz4-QWAGHFL-NH₂ (SEQ ID NO: 10) 13 L225 DO3A-monoamide-G- 240Abz4-LWAVGSFM-NH₂ (SEQ ID NO: 12) 14 L226 DO3A-monoamide-G- 5.5Abz4-HWAVGHLM-NH₂ (SEQ ID NO: 13) 15 L227 DO3A-monoamide-G- 39Abz4-LWATGHFM-NH₂ (SEQ ID NO: 17) 16 L228 DO3A-monoamide-G- 5.5Abz4-QWAVGHFM-NH₂ (SEQ ID NO: 14) 17 na GNLWATGHFM-NH₂ >500 (SEQ ID NO:20) 18 na yGNLWATGHFM-NH₂ 450 (SEQ ID NO: 20) 19 L300 DO3A-monoamide-G-2.5 Abz4-QWAVGHFL-NH₂ (SEQ ID NO: 11)

Results/Conclusions: Analysis of the binding results of various peptidesmodified in the targeting portion indicated the following:

Neuromedin analogs (GNLWATGHFM-NH₂ (SEQ ID NO: 26), yGNLWATGHFM-NH₂ (SEQID NO: 26)) are unable to compete for the GRP-R except when conjugatedto DO3A-monoamide-G-Abz4 (L227). They are, however, effective NMBcompetitors. This is similar to the requirement for derivatisation ofthe amino end of the bombesin sequence as reflected in QWAVGHLM-NH₂ (SEQID NO: 1), DO3A-monoamide-QWAVGHLM-NH₂ (SEQ ID NO: 1) & L70. Replacementof the histidine (L225) reduces competition at the GRP-R.

Reversal of the two linker components in L70 to give L204 changes thesubtype specificity to favor the NMB subtype. L¹³F substitution in thebombesin sequence maintains GRP-R activity. (L228).

TABLE 14 IC₅₀ L Number Sequence C6/NMB-R PC3/GRP-R na GNLWATGHFM-NH₂0.69 >500 (SEQ ID NO: 20) na yGNLWATGHFM-NH₂ 0.16 884.6 (SEQ ID NO: 20)L227 DO3A-monoamide-G- 0.07 28.0 Abz4-LWATGHFM-NH₂ (SEQ ID NO: 17) L225DO3A-monoamide-G- — 240 Abz4-LWAVGSFM-NH₂ (SEQ ID NO: 12) naWAVGHLM-NH₂ >800 >800 (SEQ ID NO: 19) na QWAVGHLM-NH₂ 369 754 (SEQ IDNO: 1) na DO3A-monoamide- 161 366 QWAVGHLM-NH₂ (SEQ ID NO: 1) L70DO3A-monoamide-G- 4.5 1.5 Abz4-QWAVGHLM-NH₂ (SEQ ID NO: 1) L204DO3A-monoamide-Abz4- 1.19 >50 GQWAVGHLM-NH₂ (SEQ ID NO: 22) L228DO3A-monoamide-G- — 5.5 Abz4-QWAVGHFM-NH₂ (SEQ ID NO: 14)

As seen here, F¹³M¹⁴ to F¹³L¹⁴ substitution in L228 produces a compound(L300) with high activity at the GRP-R. The removal of the methioninehas advantages as it is prone to oxidation. This benefit does not occurif the L¹³F substitution is not also performed (L221). Removal of V¹⁰resulted in complete loss of binding as seen in L224.

TABLE 15 IC50 Number Sequence C6/NMB-R PC3/GRP-R L300 DO3A-monoamide-G-— 2.5 Abz4-QWAVGHFL-NH₂ (SEQ ID NO: 11) L221 DO3A-monoamide--G- — 340Abz4-fQWAVGHLL-NH₂ (SEQ ID NO: 8) L224 DO3A-monoamide-G- — >500 Abz4-QWAGHFL-NH₂ (SEQ ID NO: 10)

TABLE 16 As seen in Table 16, various substitutions are allowed in theBBN²⁻⁶ region (L214-L217, L226) IC50 Number Sequence C6/NMB-R PC3/GRP-Rna pEQRYGNQWAVGHLM-NH₂ 3.36 2.2 (SEQ ID NO: 23) L214DO3A-monoamide-G-Abz4- — 18 fQWAVGHLM-NH₂ (SEQ ID NO: 1) L215DO3A-monoamide-G-Abz4- — 6 QRLGNQWAVGHLM-NH₂ (SEQ ID NO: 3) L216DO3A-monoamide-G-Abz4- — 4.5 QRYGNQWAVGHLM-NH₂ (SEQ ID NO: 4) L217DO3A-monoamide-G-Abz4- — 10 QKYGNQWAVGHLM-NH₂ (SEQ ID NO: 5) L226DO3A-monoamide-G-Abz4- — 5.5 HWAVGHLM-NH₂ (SEQ ID NO: 13)

TABLE 17 As expected, results from Table 17 show that the universalagonists (L222 & L223) compete reasonably well at ~50 nM level. IC50Num- C6/ PC3/ Name ber Sequence NMB-R GRP-R Universal L222DO3A-monoamide-G-Abz4- — 46 agonist yQWAV-Ala2-HF-Nle-NH₂ (SEQ ID NO: 9)Universal L224 DO3A-monoamide-G-Abz4- — 52 agonist FQWAV-Ala2-HF-Nle-NH₂(SEQ ID NO: 21)

Example LXI NMR Structural Comparison of ¹⁷⁵Lu-L70 and¹⁷⁵Lu-DO3A-Monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1)

The purpose of this NMR study was to provide complete structuralcharacterization of Lu-L70 and compare it to the structure of¹⁷⁵Lu-DOTA-Aoc-QWAVGHLM (SEQ ID NO: 1). L70 and ¹⁷⁵Lu-DOTA-Aoc-QWAVGHLM(SEQ ID NO: 1) are both bombesin analogues (see FIGS. 60 and 61),differing only in the linker between the chelating group and thetargeting peptide. In L70 there is a glycyl-4-aminobenzoyl group,whereas in ¹⁷⁵Lu-DOTA-Aoc-QWAVGHLM (SEQ ID NO: 1) there is an8-aminooctanoyl group. However, the biological data of these twocompounds is strikingly different. Detailed NMR studies were performedto explain this difference.

A. Experimental

1. Materials

5 mg of ¹⁷⁵Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH2 (SEQ ID NO: 1) wasdissolved in 225 uL of DMSO-d₆ (Aldrich 100% atom % D).

5 mg of ¹⁷⁵Lu-L70 was dissolved in 225 uL of DMSO-d₆ (Aldrich 100% atom% D).

2. Acquisition of NMR Data

All NMR experiments were performed on a Varian Inova-500 FourierTransform NMR spectrometer equipped with a 3 mm broad-band inverse(z-axis gradient) probe. The chemical shifts were referenced to theresidual CH peaks of DMSO-d₆ at 2.50 ppm for the proton and 40.19 ppmfor ¹³C. The sample temperatures were controlled by a Varian digitaltemperature controller. The data were processed using NMRPipe, VNMR,PROSA, and VNMRJ software on the Sun Blade 2000 Unix computer andanalyzed using NMRView and SPARKY software on the Linux computer. Themodeling of the peptides was performed employing CYANA software on theLinux computer and further analyzed using MOLMOL software on a CompaqDeskpro Workstation.

B. Results and Discussion

The proton chemical shifts of ¹⁷⁵Lu-L70 were assigned as follows. Aquick survey of the methyl region (0.5 to 2.5 ppm) in the 1D spectrumallowed the identification of a sharp singlet at 2.02 ppm as the methylpeak of methionine. In the same region of the TOCSY spectrum, thechemical shift at 1.16 ppm which correlates to only one peak at 4.32 ppmindicates that they belong to alanine. The methyl peaks at 0.84 and 0.85ppm which correlate to two peaks at 1.98 and 4.12 ppm must belong tovaline. The remaining methyl peaks at 0.84 and 0.88 ppm which correlateto peaks at 1.60, 1.48, and 4.23 ppm belong to leucine. These chemicalshifts and the chemical shifts of other amino acids are also present inthe “fingerprint” region (see Wuthrich, K. “NMR of Proteins and NucleicAcids”, John Wiley & Sons, 1986)—the backbone NH-αH region of the TOCSYspectrum (see FIG. 52). All the chemical shifts belonging to a spinsystem of an amino acid will align themselves vertically. After acareful examination of the spectrum, all chemical shifts were assigned.The chemical shifts were further verified by reviewing other spectrasuch as COSY (see FIG. 53) and NOESY (see FIG. 54). After the protonchemical shifts were assigned, their carbon chemical shifts wereidentified through the gHSQC spectrum (see FIG. 55) and further verifiedby reviewing the gHMBC (see FIG. 56) and gHSQCTOCSY (see FIG. 57)spectra. The chemical shifts of Lu-L70 are listed in Table 19 (the atomnumbers are referenced to FIG. 60).

Interestingly, in the TOCSY spectrum of ¹⁷⁵Lu-L70, the chemical shift ofthe NH proton at 14.15 ppm shows strong correlations to two other peaksof the histidine ring, and also to a water molecule. This water moleculeis not freely exchanging and is clearly seen in the NMR timeframe. Tosee which proton of the histidine interacts more strongly with the watermolecule, a selective homo-decoupling experiment was performed on the¹⁷⁵Lu-L70 at 15° C. When the water peak was selectively saturated with alow power, the intensities of the NH peaks of histidine at 14.16 and14.23 ppm were dramatically reduced while the intensities of the tworemaining peaks of histidine at 7.32 and 8.90 ppm were partially reduced(see FIG. 58). The observation of the water protons on the NMR timescale suggests a rigid confirmation.

A proposed chemical structure of ¹⁷⁵Lu-L70 with a water molecule can beseen in FIG. 62. A water molecule occupies a ninth coordination site bycapping the square plane described by the coordinated oxygens. This hasother precedents. Coordination of water at the ninth site of Lu inNa[Lu(DOTA)H₂O)].4H₂O was observed in an x-ray structure, as shown byAime et al, Inorg. Chim. Acta 1996, 246, 423-429, which is incorporatedby reference.

In contrast, in the TOCSY spectrum of¹⁷⁵Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂ (SEQ ID NO: 1), the chemical shiftof the NH proton only shows strong correlations to two other peaks ofthe histidine ring, but not to the water molecule (see FIG. 59). Thisindicates that there is no water molecule simultaneously coordinatingboth the ¹⁷⁵Lu and the His-NH in ¹⁷⁵Lu-DO3A-monoamide-Aoc-QWAVGHLM-NH₂(SEQ ID NO: 1). Thus, the difference between the two molecules issignificant. In the ¹⁷⁵Lu-L70 a secondary structure of the peptide isstabilized via the bound water molecule, and this may be responsible forincreased in vivo stability.

TABLE 19 Chemical Shifts (ppm) of ¹⁷⁵Lu-L70 in DMSO-d₆ at 25 °C.Position Chemical Shift Assignment Proton (Carbon)  2/12 —  3/11 —  5/9—  6/8 — 13 — 15 — 20 — 17 3.69/3.62 22 9.95/9.73 23 4.04/4.16 (43.57)26 10.47 28/32 7.62 (118.9) 29/31 7.79 (128.7) 35a 8.54 36 4.29 (54.26)39 1.83/1.91 (27.26) 40 2.16 (32.08) 47 6.84/7.30 43 7.97 44 4.54(53.37) 48 2.98/3.12 (27.74) 50 7.12 (123.9) 51 10.79 53 7.53 (118.7) 546.93 (118.6) 55 7.03 (121.3) 56 7.28 (111.7) 58 8.09 59 4.32 (48.71) 621.16 (17.86) 63 7.65 64 4.12 (58.28) 67 1.98 (30.96) 68/73 0.84 (18.42)0.85 (19.52) 69 8.19 70 3.70/3.74 (42.45) 74 8.10 75 4.60 (51.85) 782.95/3.08 (27.50) 80 14.15 81 8.91 83 7.32 84 8.14 85 4.23 (51.93) 861.48 (40.6) 87 1.60 (24.61) 88/91 0.84 (21.8) 0.88 (23.41) 92 8.04 934.25 (52.25) 96 1.76/1.92 (32.16) 97 2.41 (29.91) 99 2.02 (15.13)

1. A compound of the general formula:M—N—O—P—G wherein M is an optical label or a metal chelator optionallycomplexed with a radionuclide; N is absent, an alpha amino acid, anon-alpha amino acid with a cyclic group or other linking group; O is analpha amino acid or a non-alpha amino acid with a cyclic group; P isabsent, an alpha amino acid, a non-alpha amino acid with a cyclic group,or other linking group; and G is a GRP receptor targeting peptideselected from the group consisting of QWAVGHLM-OH (SEQ ID NO: 1),QWAVGHLM-NH₂ (SEQ ID NO: 1), QWAVGHFL-NH₂ (SEQ ID NO: 11),QRLGNQWAVGHLM-NH₂ (SEQ ID NO: 3), QRYGNQWAVGHLM-NH₂ (SEQ ID NO: 4),QKYGNQWAVGHLM-NH₂ (SEQ ID NO: 5), QWAVGHL-NH-Pentyl (SEQ ID NO: 6),QWSVaHLM-NH₂ (SEQ ID NO: 7), QWAVGHLL-NH₂ (SEQ ID NO: 8),QWAV-Bala-HF-Nle-NH₂ (SEQ ID NO: 9), QWAGHFL-NH₂ (SEQ ID NO: 10),LWAVGSFM-NH₂ (SEQ ID NO: 12), HWAVGHLM-NH₂ (SEQ ID NO: 13), LWATGHFM-NH₂(SEQ ID NO: 17), LWAVGSFM-NH₂ (SEQ ID NO: 12), EWAVGHLM-NH₂ (SEQ ID NO:2), QWAVaHLM-NH₂ (SEQ ID NO: 15), QWAVGHFM-NH₂ (SEQ ID NO: 14),Nme-QWAVGHLM-NH₂ (SEQ ID NO: 1), Q-Ψ[CSNH]WAVGHLM-NH₂ (SEQ ID NO: 1),Q-Ψ[CH₂NH]-WAVGHLM-NH₂ (SEQ ID NO: 1), Q-Ψ[CH═CH]WAVGHLM-NH₂ (SEQ ID NO:1), α-MeQWAVGHLM-NH₂ (SEQ ID NO: 24), QNme-WAVGHLM-NH₂ (SEQ ID NO: 29),QW-Ψ[CSNH]-AVGHLM-NH₂ (SEQ ID NO: 1), QW-Ψ[CH₂NH]-AVGHLM-NH₂ (SEQ ID NO:1), QW-Ψ[CH═CH]-AVGHLM-NH₂ (SEQ ID NO: 1), Q-α-Me-WAVGHLM-NH₂ (SEQ IDNO: 30), QW-Nme-AVGHLM-NH₂ (SEQ ID No: 31), QWA=Ψ[CSNH]-VGHLM-NH₂ (SEQID NO: 1), QWA-Ψ[CH₂NH]-VGHLM-NH₂ (SEQ ID No: 1), QW-Aib-VGHLM-NH₂ (SEQID NO: 1), QWAV-Sar-HLM-NH₂ (SEQ ID No: 32), QWAVG-Ψ[CSNH]-HLM-NH₂ (SEQID NO: 1), QWAVG-Ψ[CH═CH]-HLM-NH₂ (SEQ ID NO: 1), QWAV-Dala-HLM-NH₂ (SEQID NO: 15), QWAVG-Nme-His-LM-NH₂ (SEQ ID NO: 33),QWAVG-H-Ψ[CSNH]-L-M-NH₂ (SEQ ID NO: 1), QWAVG-H-Ψ[CH₂NH]-LM-NH₂ (SEQ IDNO: 1), QWAVGH-Ψ[CH═CH]-LM-NH₂ (SEQ ID NO: 1), QWAVG-α-Me-HLM-NH₂ (SEQID NO: 34), QWAVGH-Nme-LM-NH₂ (SEQ ID NO: 35), and QWAVGH-α-MeLM-NH₂(SEQ ID NO: 28), wherein at least one of N, O or P is a non-alpha aminoacid with a cyclic group and wherein the other linking group of N or Pis selected from the group consisting of one or more amino acids, ahydrocarbon chain of the formula R₁—(CH₂)_(n)—R₂ or a combinationthereof, wherein n is 0-10, R₁ is selected from the group consisting ofH₂N—, HS— and —COOH; and R₂ is COOH.
 2. The compound of claim 1, whereinthe non-alpha amino acid with a cyclic group is selected from the groupconsisting of: 4-aminobenzoic acid; 4-aminomethyl benzoic acid;trans-4-aminomethylcyclohexane carboxylic acid; 4-(2-aminoethoxy)benzoicacid; isonipecotic acid; 2-aminomethylbenzoic acid;4-amino-3-nitrobenzoic acid;4-(3-carboxymethyl-2-keto-1-benzimidazolyl)-piperidine;6-(piperazin-1-yl)-4-(3H)-quinazolinone-3-acetic acid;(2s,5s)-5-amino-1,2,4,5,6,7-hexahydro-4-oxo-azepino[3,2,1-hi]indole-2-carboxylicacid,(4S,7R)-4-amino-6-aza-5-oxo-9-thiabicyclo[4.3.0]nonane-7-carboxylicacid; 3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one;N1-piperazineacetic acid; N-4-aminoethyl-N-1-acetic acid;(3S)-3-amino-1-carboxymethylcaprolactam; and(2S,6S,9)-6-amino-2-carboxymethyl-3,8-diazabicyclo-[4,3,0]-nonane-1,4-dione;1-naphthylalanine; 3′-aminomethyl-biphenyl-3-carboxylic acid;4-aminomethylphenoxyacetic acid; 4-aminophenylacetic acid; 4-phenoxy;3-aminomethylbenzoic acid; 4-aminomethyl-3-methoxybenzoic acid;4-hydrazinobenzoyl; 6-aminonicotinic acid;4-amino-2′-methylbiphenyl-4-carboxylic acid; Terephthalic acid;3-aminobenzoic acid; 6-aminonaphthoic acid; 3-amino-3-deoxycholoic acid;3-methoxy-4-aminobenzoic acid; 3-chloro-4-aminobenzoic acid; and3-hydroxy-4-aminobenzoic acid.
 3. The compound of claim 1, wherein M isselected from the group consisting of: DTPA, DOTA, DO3A, HPDO3A, EDTA,and TETA.
 4. The compound of claim 1, wherein M is selected from thegroup consisting of EHPG, 5-Cl-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG,and 5-sec-Bu-EHPG.
 5. The compound of claim 1, wherein M is selectedfrom the group consisting of benzodiethylenetriamine pentaacetic acid(benzo-DTPA), dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, anddibenzyl DTPA.
 6. The compound of claim 1, wherein M is selected fromthe group consisting of HBED.
 7. The compound of claim 1, wherein M isselected from the group consisting of benzo-DOTA, dibenzo-DOTA, andbenzo-NOTA, benzo-TETA, benzo-DOTMA, and benzo-TETMA.
 8. The compound ofclaim 1, wherein M is selected from the group consisting of1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA);1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). 9.The compound of claim 1, selected from the group consisting of:DO3A-monoamide-G-4-aminobenzoic acid-EWAVGHLM-NH₂ (SEQ ID NO: 2);DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHLM-OH (SEQ ID NO: 1);DO3A-monoamide-G-4-aminobenzoic acid-(D)-Phe-BBN(7-14);DO3A-monoamide-G-4-aminobenzoic acid-QRLGNQWAVGHLM-NH₂ (SEQ ID NO: 3);DO3A-monoamide-G-4-aminobenzoic acid-QRYGNQWAVGHLM-NH₂ (SEQ ID NO: 4);DO3A-monoamide-G-4-aminobenzoic acid-QKYGNQWAVGHLM-NH₂ (SEQ ID NO: 5);DO3A-monoamide-G-4-aminobenzoic acid-(D)-Phe-QWAVGHL-NH-Pentyl (SEQ IDNO: 6); DO3A-monoamide-G-4-aminobenzoic acid-QWSVaHLM-NH₂ (SEQ ID NO:7); DO3A-monoamide-G-4-aminobenzoic acid-(D)-Phe-QWAVGHLL-NH₂ (SEQ IDNO: 8); DO3A-monoamide-G-4-aminobenzoicacid-(D)-Tyr-QWAV-Bala-HF-Nle-NH₂ (SEQ ID NO: 9);DO3A-monoamide-G-4-aminobenzoic acid-Phe-QWAV-Bala-HF-Nle-NH₂ (SEQ IDNO: 9); DO3A-monoamide-G-4-aminobenzoic acid-QWAGHFL-NH₂ (SEQ ID NO:10); DO3A-monoamide-G-4-aminobenzoic acid-LWAVGSFM-NH₂ (SEQ ID NO: 12);DO3A-monoamide-G-4-aminobenzoic acid-HWAVGHLM-NH₂ (SEQ ID NO: 13);DO3A-monoamide-G-4-aminobenzoic acid-LWAVGSFM-NH₂ (SEQ ID NO: 12);DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHFM-NH₂ (SEQ ID NO: 14);DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHFL-NH₂ (SEQ ID NO: 11);DO3A-monoamide-4-aminomethylbenzoicacid-L-1-Naphthylalanine-QWAVGHLM-NH2 (SEQ ID NO: 1); andDO3A-monoamide-G-4-aminobenzoic acid-QWAVGNMeHisLM-NH₂ (SEQ ID NO: 16).10. The compound of any one of claims 1 or 2, wherein the optical labelis selected from the group consisting of organic chromophores, organicfluorophores, light-absorbing compounds, light-reflecting compounds,light-scattering compounds, and bioluminescent molecules.
 11. A methodof imaging a patient comprising the steps of: administering to a subjecta diagnostic imaging agent comprising the compound of claim 1 wherein Mis a metal chelator complexed with a diagnostic radionuclide, andimaging said patient.
 12. A method of imaging a patient comprising thesteps of: administering to a patient a diagnostic imaging agentcomprising the compound of claim 9, and imaging said patient.
 13. Amethod of imaging a patient comprising the steps of: administering to apatient a diagnostic imaging agent comprising the compound of claim 1,wherein M is an optical label, and imaging said patient.
 14. A methodfor preparing a diagnostic imaging agent comprising the step of addingto an injectable medium a substance comprising the compound of claim 1.15. A method of treating a patient in need of radiotherapy comprisingthe step of administering to a patient a radiotherapeutic agentcomprising the compound of claim 1 complexed with a therapeuticradionuclide.
 16. A method of preparing a radiotherapeutic agentcomprising the step of adding to an injectable medium a substancecomprising the compound of claim
 1. 17. A compound of the generalformula:M—N—O—P—G wherein M is DO3A, optionally complexed with a radionuclide; Nis absent, an alpha or non-alpha amino acid or other linking group; O isan alpha or non-alpha amino acid; and P is absent, an alpha or non-alphaamino acid or other linking group, and G is a GRP receptor targetingpeptide selected from the group consisting of QWAVGHLM-OH (SEQ ID NO:1), QWAVGHLM-NH₂ (SEQ ID NO: 1), QWAVGHFL-NH₂ (SEQ ID NO: 11),QRLGNQWAVGHLM-NH₂ (SEQ ID NO: 3), QRYGNQWAVGHLM-NH₂ (SEQ ID NO: 4),QKYGNQWAVGHLM-NH₂ (SEQ ID NO: 5), QWAVGHL-NH-Pentyl (SEQ ID NO: 6),QWSVaHLM-NH₂ (SEQ ID NO: 7), QWAVGHLL-NH₂ (SEQ ID NO: 8),QWAV-Bala-HF-Nle-NH₂ (SEQ ID NO: 9), QWAGHFL-NH₂ (SEQ ID NO: 10),LWAVGSFM-NH₂ (SEQ ID NO: 12), HWAVGHLM-NH₂ (SEQ ID NO: 13), LWATGHFM-NH₂(SEQ ID NO: 17), LWAVGSFM —NH₂ (SEQ ID NO: 12), EWAVGHLM-NH₂ (SEQ ID NO:2), QWAVaHLM —NH₂ (SEQ ID NO: 15), QWAVGHFM-NH₂ (SEQ ID NO: 14),Nme-QWAVGHLM-NH₂ (SEQ ID NO: 1), Q-Ψ[CSNH]WAVGHLM-NH₂ (SEQ ID NO: 1),Q-Ψ[CH₂NH]-WAVGHLM-NH₂ (SEQ ID NO: 1), Q-Ψ[CH═CH]WAVGHLM-NH₂ (SEQ ID NO:1), α-MeQWAVGHLM-NH₂ (SEQ ID NO: 24), QNme-WAVGHLM-NH₂ (SEQ ID NO: 29),QW-Ψ[CSNH]-AVGHLM-NH₂ (SEQ ID NO: 1), QW-Ψ[CH₂NH]-AVGHLM-NH₂ (SEQ ID NO:1), QW-Ψ[CH═CH]-AVGHLM-NH₂ (SEQ ID NO: 1), Q-α-Me-WAVGHLM-NH₂ (SEQ IDNO: 30), QW-Nme-AVGHLM-NH₂ (SEQ ID NO: 31), QWA=Ψ[CSNH]-VGHLM-NH₂ (SEQID NO: 1), QWA-Ψ[CH₂NH]-VGHLM-NH₂ (SEQ ID NO: 1), QW-Aib-VGHLM-NH₂ (SEQID NO: 1), QWAV-Sar-HLM-NH₂ (SEQ ID NO: 32), QWAVG-Ψ[CSNH]-HLM-NH₂ (SEQID NO: 1), QWAVG-Ψ[CH═CH]-HLM-NH₂ (SEQ ID NO: 1), QWAV-Dala-HLM-NH₂ (SEQID NO: 15), QWAVG-Nme-His-LM-NH₂ (SEQ ID NO: 33),QWAVG-H-Ψ[CSNH]-L-M-NH₂ (SEQ ID No: 1), QWAVG-H-Ψ[CH₂NH]-LM-NH₂ (SEQ IDNO: 1), QWAVGH-Ψ[CH═CH]-LM-NH₂ (SEQ ID NO: 1), QWAVG-α-Me-HLM-NH₂ (SEQID NO: 34), QWAVGH-Nme-LM-NH₂ (SEQ ID NO: 35), and QWAVGH-α-MeLM-NH₂(SEQ ID NO: 28), wherein at least one of N, O or P is 4-aminobenzoicacid and wherein the other linking group of N or P is selected from thegroup consisting of one or more amino acids, a hydrocarbon chain of theformula R₁—(CH₂)_(n)—R₂ or a combination thereof, wherein n is 0-10, R₁is selected from the group consisting of H₂N—, HS— and —COOH; and R₂ isCOOH.
 18. A method of phototherapy of a patient in need thereofcomprising administering to a patient a compound of claim 1 wherein M isan optical label useful in phototherapy.
 19. A compound selected fromthe group consisting of: DO3A-monoamide-G-4-aminobenzoicacid-QWAVaHLM-NH₂ (SEQ ID NO: 15), DO3A-monoamide-G-4-aminobenzoicacid-fQWAVGHLM-NH₂ (SEQ ID NO: 1), DO3A-monoamide-G-4-aminobenzoicacid-fQWAVGHLL-NH₂ (SEQ ID NO: 8), DO3A-monoamide-G-4-aminobenzoicacid-fQWAVGHL-NH-pentyl (SEQ ID NO: 6), DO3A-monoamide-G-4-aminobenzoicacid-yQWAV-Bala-HFNle-NH₂ (SEQ ID NO: 9),DO3A-monoamide-G-4-aminobenzoic acid-fQWAV-Bala-HFNle-NH₂ (SEQ ID NO:9), DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHFL-NH₂ (SEQ ID NO: 11),DO3A-monoamide-G-4-aminobenzoic acid-QWAVGNMeHisLM-NH₂ (SEQ ID NO: 16),DO3A-monoamide-G-4-aminobenzoic acid-LWAVGSFM-NH₂ (SEQ ID NO: 12),DO3A-monoamide-G-4-aminobenzoic acid-HWAVGHLM-NH₂ (SEQ ID NO: 13),DO3A-monoamide-G-4-aminobenzoic acid-LWATGHFM-NH₂ (SEQ ID NO: 17),DO3A-monoamide-G-4-aminobenzoic acid-QWAVGHFM-NH₂ (SEQ ID NO: 14),DO3A-monoamide-G-4-aminobenzoic acid-QRLGNQWAVGHLM-NH₂ (SEQ ID NO: 3),DO3A-monoamide-G-4-aminobenzoic acid-QRYGNQWAVGHLM-NH₂ (SEQ ID NO: 4),DO3A-monoamide-G-4-aminobenzoic acid-QKYGNQWAVGHLM-NH₂ (SEQ ID NO: 5),Pglu-Q-Lys(DO3A-monoamide-G-4-aminobenzoic acid)-LGNQWAVGHLM-NH₂ (SEQ IDNO: 18).
 20. The method of claim 15 further comprising administering achemotherapeutic or a monoclonal antibody.
 21. A method for targetingthe gastrin releasing peptide receptor (GRP-R) and neuromedin-B receptor(NMB-R), said method comprising administering a compound of any one ofclaims 1 or
 17. 22. The method of claim 21, wherein N is Gly, O is4-aminobenzoic acid and P is absent.
 23. A compound having the followingstructure:


24. The compound of claim 1, wherein M is selected from the groupconsisting of Boa and Cm4pm10d2a.
 25. The compound of claim 1, where Mis selected from the group consisting of: N,N-dimethylGly-Ser-Cys;N,N-dimethylGly-Thr-Cys; N,N-diethylGly-Ser-Cys;N,N-dibenzylGly-Ser-Cys; N,N-dimethylGly-Ser-Cys-Gly;N,N-dimethylGly-Thr-Cys-Gly; N,N-diethylGly-Ser-Cys-Gly; andN,N-dibenzylGly-Ser-Cys-Gly.